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Xi B, An X, Yue Y, Shen H, Han G, Yang Y, Zhao S. Identification and profiling of microRNAs during sheep's testicular development. Front Vet Sci 2025; 12:1538990. [PMID: 40230794 PMCID: PMC11994653 DOI: 10.3389/fvets.2025.1538990] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2024] [Accepted: 03/06/2025] [Indexed: 04/16/2025] Open
Abstract
The normal development of the testis is essential for male reproduction, as it is the site of sperm production and a prerequisite for spermatogenesis. MiRNAs play crucial roles in various testicular biological processes, including cell proliferation, spermatogenesis, hormone secretion, metabolism, and reproductive regulation. In this study, we utilized deep sequencing data to analyze the expression patterns of small RNAs in testicular tissues of Southern × Hu sheep F1 hybrids at 0, 3, 6 months, and 1 year of age, thereby exploring the functions of miRNAs in testicular development and spermatogenesis. A total of 787 known miRNAs and 415 novel miRNAs were identified. We identified 217, 254, 405, 130, 305, and 138 DE miRNAs in the testes of M0 vs. M3, M0 vs. M6, M0 vs. Y1, M3 vs. M6, M3 vs. Y1, and M6 vs. Y1, respectively. GO annotation and KEGG pathway analysis of DE miRNA target genes revealed that target genes such as YAP1, ITGB1, DOT1L, SMAD4, and SOX9 may be involved in various biological processes, including reproductive pathways such as FOXO, Hippo, Wnt, cAMP, Rap1, and MAPK signaling pathways. The expression levels of 12 randomly selected miRNAs in testes at 0, 3, 6 months, and 1 year of age were detected by qRT-PCR, and the results were consistent with the sequencing data. This study characterized and investigated the differential expression of miRNAs in sheep testes at different developmental stages using deep sequencing technology. These findings will contribute to a deeper understanding of the functions of miRNAs in regulating testicular development and enhancing reproductive performance in male sheep.
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Affiliation(s)
- Binpeng Xi
- College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, China
| | - Xuejiao An
- Key Laboratory of Animal Genetics and Breeding on the Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou, China
- Sheep Breeding Engineering Technology Research Center of Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Yaojing Yue
- Key Laboratory of Animal Genetics and Breeding on the Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou, China
- Sheep Breeding Engineering Technology Research Center of Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Haimiao Shen
- Dongxiang County Mutton Sheep Industry Research Center, Linxia, China
| | - Gaohui Han
- Dongxiang County Animal Husbandry Development Center, Linxia, China
| | - Yanan Yang
- College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, China
| | - Shengguo Zhao
- College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, China
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2
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Moon SA, Kim JM, Lee YS, Cho HJ, Choi YJ, Yoon JH, Kim D, Che X, Jin X, Baek IJ, Lee SH, Choi JY, Koh JM. VGF and the VGF-derived peptide AQEE30 stimulate osteoblastic bone formation through the C3a receptor. Exp Mol Med 2025; 57:637-651. [PMID: 40082672 PMCID: PMC11958639 DOI: 10.1038/s12276-025-01419-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2024] [Revised: 12/05/2024] [Accepted: 12/23/2024] [Indexed: 03/16/2025] Open
Abstract
New therapeutic targets, especially those that stimulate bone formation in cortical bone, are needed to overcome the limitations of current antiosteoporotic drugs. We previously demonstrated that factors secreted from megakaryocytes (MKs) promote bone formation. Here we conducted a proteomic analysis to identify a novel bone-forming factor from MK secretions. We revealed that Vgf, a nerve growth factor-responsive gene, and its derived active peptide AQEE30 in MK-conditioned medium play important roles in osteoblast proliferation and in vitro bone formation. In both Vgf-deficient male and female mice, the cortical bone mass was significantly decreased due to reductions in osteoblast number and bone formation activity. AQEE30 stimulated intracellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity in osteoblasts, whereas an adenylyl cyclase inhibitor blocked AQEE30-stimulated osteoblast proliferation and in vitro bone formation. Complement C3a receptor-1 (C3AR1) was expressed and interacted with AQEE30 in osteoblasts, and C3AR1 inhibition blocked all AQEE30-induced changes, including stimulated proliferation, bone formation and cAMP production, in osteoblasts. Injecting mini-PEGylated AQEE30 into calvaria increased the number of osteocalcin-positive cells and new bone formation. In conclusion, this study reveals a novel role of VGF in bone formation, particularly in cortical bone, and shows that AQEE30, a VGF-derived peptide, mediates this role by activating cAMP-PKA signaling via the C3AR1 receptor in osteoblasts.
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Affiliation(s)
- Sung-Ah Moon
- Department of Medical Science, AMIST, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
| | - Jin-Man Kim
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea
| | - Young-Sun Lee
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea
| | - Han Jin Cho
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea
| | - Young Jin Choi
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea
| | - Jong Hyuk Yoon
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
- Neurodegenerative Disease Research Group, Korea Brain Research Institute, Daegu, Republic of Korea
| | - Dayea Kim
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
- New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation (K-MEDI hub), Daegu, Republic of Korea
| | - Xiangguo Che
- Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, Korea Mouse Phenotyping Center, School of Medicine, Kyungpook National University, Daegu, Republic of Korea
| | - Xian Jin
- Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, Korea Mouse Phenotyping Center, School of Medicine, Kyungpook National University, Daegu, Republic of Korea
| | - In-Jeoung Baek
- Department of Cell and Genetic Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
| | - Seung Hun Lee
- Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
| | - Je-Young Choi
- Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, Korea Mouse Phenotyping Center, School of Medicine, Kyungpook National University, Daegu, Republic of Korea.
| | - Jung-Min Koh
- Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
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Uzun S, Özcan Ö, Gök A, Işık A, Bakır S, Günel-Özcan A, Onbaşılar İ, Akyol A. A new CRISPR-mediated Apc knockout allele leads to pyloric gland adenoma-like gastric polyps in mice with C57BL/6;FVB/N mixed background. Animal Model Exp Med 2025. [PMID: 39956793 DOI: 10.1002/ame2.70002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2024] [Accepted: 01/22/2025] [Indexed: 02/18/2025] Open
Abstract
Adenomatous polyposis coli (APC) mutations are the most frequently identified genetic alteration in sporadic colorectal cancer (CRC) cases, and a myriad of genetically engineered Apc-mutant CRC mouse models have been developed using various genetic manipulation techniques. The advent of the CRISPR/Cas9 system has revolutionized the field of genetic engineering and facilitated the development of new genetically engineered mouse models. In this study, we aimed to develop a novel Apc knockout allele using the CRISPR/Cas9 system and evaluate the phenotypic effects of this new allele in two different mouse strains. For this purpose, exon 16 of mouse Apc gene was targeted with a single-guide RNA, and the mouse carrying an Apc frameshift mutation at codon 750 (Δ750) was chosen as the founder. The mutant FVB-ApcΔ750 mice were backcrossed with wild-type C57BL/6 mice, and the phenotypic effects of the knockout allele were evaluated in F8-FVB-ApcΔ750, F4-B6;FVB-ApcΔ750, and F1-B6;FVB-ApcΔ750 by a macroscopic and microscopic examination of the gastrointestinal system. The result showed that the mean polyp number was significantly higher in F4-BL6;FVB-ApcΔ750 than in F8-FVB-ApcΔ750. Intestinal polyposis was more prominent in F4-BL6;FVB-ApcΔ750, whereas a higher number of colon polyps than intestinal polyps were observed in F8-FVB-ApcΔ750. Additionally, F1-BL6;FVB-ApcΔ750 mixed background mice developed gastric polyps that morphologically resembled the pyloric gland adenoma of humans. In conclusion, we developed a novel CRISPR-mediated Apc knockout allele using two mouse strains. We showed that this allele can exert a strain-specific effect on the phenotype of mice and can cause gastric polyp formation.
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Affiliation(s)
- Sarp Uzun
- Hacettepe University Transgenic Animal Technologies Research and Application Center, Ankara, Turkey
| | - Özge Özcan
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara, Turkey
| | - Ayşenur Gök
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara, Turkey
| | - Aynur Işık
- Hacettepe University Transgenic Animal Technologies Research and Application Center, Ankara, Turkey
| | - Sinem Bakır
- Hacettepe University Transgenic Animal Technologies Research and Application Center, Ankara, Turkey
| | - Ayşen Günel-Özcan
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara, Turkey
| | - İlyas Onbaşılar
- Hacettepe University Transgenic Animal Technologies Research and Application Center, Ankara, Turkey
| | - Aytekin Akyol
- Hacettepe University Transgenic Animal Technologies Research and Application Center, Ankara, Turkey
- Department of Pathology, Hacettepe University Faculty of Medicine, Ankara, Turkey
- Tumor Pathology Division, Hacettepe University Cancer Institute, Ankara, Turkey
- Hacettepe University Molecular Pathology Research and Application Center, Ankara, Turkey
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Xie H, Linning-Duffy K, Demireva EY, Toh H, Abolibdeh B, Shi J, Zhou B, Iwase S, Yan L. CRISPR-based genome editing of a diurnal rodent, Nile grass rat (Arvicanthis niloticus). BMC Biol 2024; 22:144. [PMID: 38956550 PMCID: PMC11218167 DOI: 10.1186/s12915-024-01943-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Accepted: 06/21/2024] [Indexed: 07/04/2024] Open
Abstract
BACKGROUND Diurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. The present study aims to address this major limitation by developing experimental procedures necessary for genome editing in a well-established diurnal rodent model, the Nile grass rat (Arvicanthis niloticus). RESULTS A superovulation protocol was established, which yielded nearly 30 eggs per female grass rat. Fertilized eggs were cultured in a modified rat 1-cell embryo culture medium (mR1ECM), in which grass rat embryos developed from the 1-cell stage into blastocysts. A CRISPR-based approach was then used for gene editing in vivo and in vitro, targeting Retinoic acid-induced 1 (Rai1), the causal gene for Smith-Magenis Syndrome, a neurodevelopmental disorder. The CRISPR reagents were delivered in vivo by electroporation using an improved Genome-editing via Oviductal Nucleic Acids Delivery (i-GONAD) method. The in vivo approach produced several edited founder grass rats with Rai1 null mutations, which showed stable transmission of the targeted allele to the next generation. CRISPR reagents were also microinjected into 2-cell embryos in vitro. Large deletion of the Rai1 gene was confirmed in 70% of the embryos injected, demonstrating high-efficiency genome editing in vitro. CONCLUSION We have established a set of methods that enabled the first successful CRISPR-based genome editing in Nile grass rats. The methods developed will guide future genome editing of this and other diurnal rodent species, which will promote greater utility of these models in basic and translational research.
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Affiliation(s)
- Huirong Xie
- Transgenic and Genome Editing Facility, Institute for Quantitative Health Science & Engineering, Research Technology Support Facility, Michigan State University, East Lansing, MI, 48824, USA.
| | | | - Elena Y Demireva
- Transgenic and Genome Editing Facility, Institute for Quantitative Health Science & Engineering, Research Technology Support Facility, Michigan State University, East Lansing, MI, 48824, USA
| | - Huishi Toh
- Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, USA
| | - Bana Abolibdeh
- Transgenic and Genome Editing Facility, Institute for Quantitative Health Science & Engineering, Research Technology Support Facility, Michigan State University, East Lansing, MI, 48824, USA
| | - Jiaming Shi
- Department of Psychology, Michigan State University, East Lansing, MI, 48824, USA
| | - Bo Zhou
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, USA
- Department of Pediatrics, University of Michigan Medical School, Ann Arbor, USA
| | - Shigeki Iwase
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, USA
- Department of Pediatrics, University of Michigan Medical School, Ann Arbor, USA
| | - Lily Yan
- Department of Psychology, Michigan State University, East Lansing, MI, 48824, USA.
- Neuroscience Program, Michigan State University, East Lansing, USA.
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5
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Bisht D, Salave S, Desai N, Gogoi P, Rana D, Biswal P, Sarma G, Benival D, Kommineni N, Desai D. Genome editing and its role in vaccine, diagnosis, and therapeutic advancement. Int J Biol Macromol 2024; 269:131802. [PMID: 38670178 DOI: 10.1016/j.ijbiomac.2024.131802] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2023] [Revised: 02/25/2024] [Accepted: 03/15/2024] [Indexed: 04/28/2024]
Abstract
Genome editing involves precise modification of specific nucleotides in the genome using nucleases like CRISPR/Cas, ZFN, or TALEN, leading to increased efficiency of homologous recombination (HR) for gene editing, and it can result in gene disruption events via non-homologous end joining (NHEJ) or homology-driven repair (HDR). Genome editing, particularly CRISPR-Cas9, revolutionizes vaccine development by enabling precise modifications of pathogen genomes, leading to enhanced vaccine efficacy and safety. It allows for tailored antigen optimization, improved vector design, and deeper insights into host genes' impact on vaccine responses, ultimately enhancing vaccine development and manufacturing processes. This review highlights different types of genome editing methods, their associated risks, approaches to overcome the shortcomings, and the diverse roles of genome editing.
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Affiliation(s)
- Deepanker Bisht
- ICAR- Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, India
| | - Sagar Salave
- National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad 382355, Gujarat, India
| | - Nimeet Desai
- Indian Institute of Technology Hyderabad, Kandi 502285, Telangana, India
| | - Purnima Gogoi
- School of Medicine and Public Health, University of Wisconsin and Madison, Madison, WI 53726, USA
| | - Dhwani Rana
- National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad 382355, Gujarat, India
| | - Prachurya Biswal
- College of Veterinary and Animal Sciences, Bihar Animal Sciences University, Kishanganj 855115, Bihar, India
| | - Gautami Sarma
- College of Veterinary & Animal Sciences, G. B. Pant University of Agriculture and Technology, Pantnagar 263145, U.S. Nagar, Uttarakhand, India
| | - Derajram Benival
- National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad 382355, Gujarat, India.
| | | | - Dhruv Desai
- School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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6
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Lee JG, Yon JM, Kim G, Lee SG, Kim CY, Cheong SA, Kim HY, Yu J, Kim K, Sung YH, Yoo HJ, Woo DC, Rho JK, Ha CH, Pack CG, Oh SH, Lim JS, Han YM, Hong EJ, Seong JK, Lee HW, Lee SW, Lee KU, Kim CJ, Nam SY, Cho YS, Baek IJ. PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development. Nat Commun 2024; 15:1487. [PMID: 38374152 PMCID: PMC10876648 DOI: 10.1038/s41467-024-45647-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2023] [Accepted: 01/30/2024] [Indexed: 02/21/2024] Open
Abstract
Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs, such as the heart, is crucial for a normal pregnancy. However, the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT), which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells, promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover, in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis, including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo.
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Affiliation(s)
- Jong Geol Lee
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Korea Mouse Phenotyping Center (KMPC), Seoul, 08826, Korea
- Biological Resources Research Group, Bioenvironmental Science & Toxicology Division, Gyeongnam Branch Institute, Korea Institute of Toxicology (KIT), Jinju, 52834, Korea
| | - Jung-Min Yon
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Cell and Genetic Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Globinna Kim
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Cell and Genetic Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Seul-Gi Lee
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, 05029, Korea
| | - C-Yoon Kim
- College of Veterinary Medicine, Konkuk University, Seoul, 05029, Korea
| | - Seung-A Cheong
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
| | | | - Jiyoung Yu
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
| | - Kyunggon Kim
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Digital Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Young Hoon Sung
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Cell and Genetic Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Hyun Ju Yoo
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Digital Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Dong-Cheol Woo
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Biomedical Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Jin Kyung Rho
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Chang Hoon Ha
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Chan-Gi Pack
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Biomedical Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Seak Hee Oh
- Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Joon Seo Lim
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
| | - Yu Mi Han
- Research Institute of Medical Science, Sungkyunkwan University School of Medicine, Seoul, 06351, Korea
| | - Eui-Ju Hong
- College of Veterinary Medicine, Chungnam National University, Daejeon, 34134, Korea
| | - Je Kyung Seong
- Korea Mouse Phenotyping Center (KMPC), Seoul, 08826, Korea
- College of Veterinary Medicine, Seoul National University, Seoul, 08826, Korea
| | - Han-Woong Lee
- Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Korea
| | - Sang-Wook Lee
- Korea Mouse Phenotyping Center (KMPC), Seoul, 08826, Korea
- Department of Radiation Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Ki-Up Lee
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Chong Jai Kim
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
- Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
| | - Sang-Yoon Nam
- College of Veterinary Medicine, Chungbuk National University, Cheongju, 28644, Korea
| | - You Sook Cho
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea.
- Division of Allergy and Clinical Immunology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea.
| | - In-Jeoung Baek
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea.
- Korea Mouse Phenotyping Center (KMPC), Seoul, 08826, Korea.
- Department of Cell and Genetic Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea.
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Liu R, Shang W, Liu Y, Xie Y, Luan J, Zhang T, Ma Y, Wang Z, Sun Y, Song X, Han F. Inhibition of the ILK-AKT pathway by upregulation of PARVB contributes to the cochlear cell death in Fascin2 gene knockout mice. Cell Death Discov 2024; 10:89. [PMID: 38374196 PMCID: PMC10876960 DOI: 10.1038/s41420-024-01851-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Revised: 01/29/2024] [Accepted: 02/06/2024] [Indexed: 02/21/2024] Open
Abstract
The Fscn2 (Fascin2) gene encodes an actin cross-linking protein that is involved in the formation of hair cell stereocilia and retina structure. Mutations in Fscn2 gene have been linked to hearing impairment and retinal degeneration in humans and mice. To understand the function of the Fscn2 gene, we generated the Fscn2 knockout mice, which showed progressive loss of hearing and hair cells. Our goal of the present study was to investigate the mechanism underlying cochlear cell death in the Fscn2 knockout mice. Microarray analysis revealed upregulation of expression of PARVB, a local adhesion protein, in the inner ears of Fscn2 knockout mice at 8 weeks of age. Further studies showed increased levels of PARVB together with cleaved-Caspase9 and decreased levels of ILK, p-ILK, p-AKT, and Bcl-2 in the inner ears of Fscn2 knockout mice of the same age. Knockdown of Fscn2 in HEI-OCI cells led to decreased cell proliferation ability and migration rate, along with increased levels of PARVB and decreased levels of ILK, p-ILK, p-AKT, Bcl-2 and activated Rac1 and Cdc42. Overexpression of Fscn2 or inhibition of Parvb expression in HEI-OC1 cells promoted cell proliferation and migration, with increased levels of ILK, p-ILK, p-AKT, and Bcl-2. Finally, FSCN2 binds with PPAR-γ to reduce its nuclear translocation in HEI-OC1 cells, and inhibition of PPAR-γ by GW9662 decreased the level of PARVB and increased the levels of p-AKT, p-ILK, and Bcl-2. Our results suggest that FSCN2 negatively regulates PARVB expression by inhibiting the entry of PPAR-γ into the cell nucleus, resulting in inhibition of ILK-AKT related pathways and of cochlear cell survival in Fscn2 knockout mice. Our findings provide new insights and ideas for the prevention and treatment of genetic hearing loss.
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Affiliation(s)
- Rongrong Liu
- Department of Biochemistry and Molecular Biology, and Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China
- Department of Otorhinolaryngology-Head and Neck Surgery, Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, 264000, PR China
| | - Wenjing Shang
- Department of Biochemistry and Molecular Biology, and Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China
| | - Yingying Liu
- Department of Biochemistry and Molecular Biology, and Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China
| | - Yi Xie
- Department of Biochemistry and Molecular Biology, and Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China
| | - Jun Luan
- Department of Biochemistry and Molecular Biology, and Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China
| | - Ting Zhang
- Department of Biochemistry and Molecular Biology, and Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China
| | - Ying Ma
- Department of Biochemistry and Molecular Biology, and Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China
| | - Zengxian Wang
- Institute of Neurobiology, School of Medicine, Xi'an Siyuan University, 28 Shui An Road, Xi'an, 710038, Shaanxi, PR China
| | - Yan Sun
- Department of Otorhinolaryngology-Head and Neck Surgery, Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, 264000, PR China.
| | - Xicheng Song
- Department of Otorhinolaryngology-Head and Neck Surgery, Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, 264000, PR China.
| | - Fengchan Han
- Department of Biochemistry and Molecular Biology, and Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China.
- Institute of Neurobiology, School of Medicine, Xi'an Siyuan University, 28 Shui An Road, Xi'an, 710038, Shaanxi, PR China.
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8
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Khademi Z, Mahmoudi Z, Sukhorukov VN, Jamialahmadi T, Sahebkar A. CRISPR/Cas9 Technology: A Novel Approach to Obesity Research. Curr Pharm Des 2024; 30:1791-1803. [PMID: 38818919 DOI: 10.2174/0113816128301465240517065848] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2023] [Revised: 04/11/2024] [Accepted: 04/17/2024] [Indexed: 06/01/2024]
Abstract
Gene editing technology, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has transformed medical research. As a newly developed genome editing technique, CRISPR technology has strongly assisted scientists in enriching their comprehension of the roles of individual genes and their influences on a vast spectrum of human malignancies. Despite considerable progress in elucidating obesity's molecular pathways, current anti-obesity medications fall short in effectiveness. A thorough understanding of the genetic foundations underlying various neurobiological pathways related to obesity, as well as the neuro-molecular mechanisms involved, is crucial for developing effective obesity treatments. Utilizing CRISPR-based technologies enables precise determination of the roles of genes that encode transcription factors or enzymes involved in processes, such as lipogenesis, lipolysis, glucose metabolism, and lipid storage within adipose tissue. This innovative approach allows for the targeted suppression or activation of genes regulating obesity, potentially leading to effective weight management strategies. In this review, we have provided a detailed overview of obesity's molecular genetics, the fundamentals of CRISPR/Cas9 technology, and how this technology contributes to the discovery and therapeutic targeting of new genes associated with obesity.
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Affiliation(s)
- Zahra Khademi
- Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
| | - Zahra Mahmoudi
- Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran
| | - Vasily N Sukhorukov
- Institute of General Pathology and Pathophysiology, The Russian Academy of Medical Sciences, 8 Baltiiskaya Street, Moscow 125315, Russia
| | - Tannaz Jamialahmadi
- Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
- Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Amirhossein Sahebkar
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
- Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
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9
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Sakai Y, Okabe Y, Itai G, Shiozawa S. An efficient evaluation system for factors affecting the genome editing efficiency in mouse. Exp Anim 2023; 72:526-534. [PMID: 37407493 PMCID: PMC10658088 DOI: 10.1538/expanim.23-0045] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Accepted: 06/23/2023] [Indexed: 07/07/2023] Open
Abstract
Genome editing technology is widely used in the field of laboratory animal science for the production of genetic disease models and the analysis of gene function. One of the major technical problems in genome editing is the low efficiency of precise knock-in by homologous recombination compared to simple knockout via non-homologous end joining. Many studies have focused on this issue, and various solutions have been proposed; however, they have yet to be fully resolved. In this study, we established a system that can easily determine the genotype at the mouse (Mus musculus) Tyr gene locus for genome editing both in vitro and in vivo. In this genome editing system, by designing the Cas9 cleavage site and donor template, wild-type, knockout, and knock-in genotypes can be distinguished by restriction fragment length polymorphisms of PCR products. Moreover, the introduction of the H420R mutation in tyrosinase allows the determination of knock-in mice with specific coat color patterns. Using this system, we evaluated the effects of small-molecule compounds on the efficiency of genome editing in mouse embryos. Consequently, we successfully identified a small-molecule compound that improves knock-in efficiency in genome editing in mouse embryos. Thus, this genome editing system is suitable for screening compounds that can improve knock-in efficiency.
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Affiliation(s)
- Yusuke Sakai
- Institute for Disease Modeling, Kurume University School of Medicine, 67 Asahimachi, Kurume city, Fukuoka 830-0011, Japan
| | - Yuri Okabe
- Institute for Disease Modeling, Kurume University School of Medicine, 67 Asahimachi, Kurume city, Fukuoka 830-0011, Japan
| | - Gen Itai
- Center for Integrated Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
- JAC Inc., 1-2-7 Higashiyama, Meguro-ku, Tokyo 153-0043, Japan
| | - Seiji Shiozawa
- Institute for Disease Modeling, Kurume University School of Medicine, 67 Asahimachi, Kurume city, Fukuoka 830-0011, Japan
- Center for Integrated Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
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10
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Elizalde MJ, Gorelick DA. Mechanistic toxicology in light of genetic compensation. Toxicol Sci 2023; 197:kfad113. [PMID: 37941503 PMCID: PMC10823772 DOI: 10.1093/toxsci/kfad113] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2023] Open
Abstract
Mechanistic toxicology seeks to identify the molecular and cellular mechanisms by which toxicants exert their deleterious effects. One powerful approach is to generate mutations in genes that respond to a particular toxicant, and then test how such mutations change the effects of the toxicant. CRISPR is a rapid and versatile approach to generate mutations in cultured cells and in animal models. Many studies use CRISPR to generate short insertions or deletions in a target gene and then assume that the resulting mutation, such as a premature termination codon, causes a loss of functional protein. However, recent studies demonstrate that this assumption is flawed. Cells can compensate for short insertion and deletion mutations, leading toxicologists to draw erroneous conclusions from mutant studies. In this review, we will discuss mechanisms by which a mutation in one gene may be rescued by compensatory activity. We will discuss how CRISPR insertion and deletion mutations are susceptible to compensation by transcriptional adaptation, alternative splicing, and rescue by maternally derived gene products. We will review evidence that measuring levels of messenger RNA transcribed from a mutated gene is an unreliable indicator of the severity of the mutation. Finally, we provide guidelines for using CRISPR to generate mutations that avoid compensation.
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Affiliation(s)
- Mary Jane Elizalde
- Department of Molecular & Cellular Biology, Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030, United States
| | - Daniel A Gorelick
- Department of Molecular & Cellular Biology, Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030, United States
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11
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Wadbudhe AM, Meshram RJ, Tidke SC. Severe Combined Immunodeficiency (SCID) and Its New Treatment Modalities. Cureus 2023; 15:e47759. [PMID: 38022338 PMCID: PMC10676291 DOI: 10.7759/cureus.47759] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2023] [Accepted: 10/26/2023] [Indexed: 12/01/2023] Open
Abstract
Severe combined immunodeficiency (SCID) is a rare condition with very high mortality. SCID is mainly caused by the multiple mutations of genes affecting the entire immune cells. Children with this disease are born with an impaired immune system. The child appears healthy but the consequences of the impaired immune system lead to various secondary infections such as meningeal infections and respiratory infections further leading to consolidation, diarrhea, inflammation of skin and other systemic diseases. Severe combined immunodeficiency is also known as "bubble boy disease" or "living in the bubble" syndrome, as in early days for treatment the physicians decided to completely isolate them until they got the perfect match for the bone marrow transplantation. It is one of the pediatric emergencies and is to be treated as soon as possible. SCID involves multiple genes which leads to makes diagnosis of the disease cumbersome. In early years many infants were diagnosed almost after half a year and in severe conditions which led to the decrease in the survival rate of the children. But now due to advanced newborn screening modalities and other monitoring systems it can be diagnosed as early as within three months of age. The various treatment modalities include hematopoietic stem cell transplantation, gene therapy, enzyme replacement therapy and chemotherapy. This narrative review article describes about the severe combined immunodeficiency and its newer treatment modalities.
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Affiliation(s)
- Akshad M Wadbudhe
- Department of Paediatrics, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
| | - Revat J Meshram
- Department of Paediatrics, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
| | - Shivangi C Tidke
- Department of Paediatrics, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
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12
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Xie H, Linning-Duffy K, Demireva EY, Toh H, Abolibdeh B, Shi J, Zhou B, Iwase S, Yan L. CRISPR-based Genome Editing of a Diurnal Rodent, Nile Grass Rat ( Arvicanthis niloticus). BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.23.553600. [PMID: 37662225 PMCID: PMC10473663 DOI: 10.1101/2023.08.23.553600] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2023]
Abstract
Diurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. Here, we address this major limitation by demonstrating the first successful CRISPR genome editing of the Nile grass rat ( Arvicanthis niloticus ), a valuable diurnal rodent. We establish methods for superovulation; embryo development, manipulation, and culture; and pregnancy maintenance to guide future genome editing of this and other diurnal rodent species.
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13
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Wang J, Gao Y, Xiao L, Lin Y, Huang L, Chen J, Liang G, Li W, Yi W, Lao J, Zhang B, Gao TM, Zhong M, Yang X. Increased NMDARs in neurons and glutamine synthetase in astrocytes underlying autistic-like behaviors of Gabrb1-/- mice. iScience 2023; 26:107476. [PMID: 37599823 PMCID: PMC10433130 DOI: 10.1016/j.isci.2023.107476] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Revised: 06/16/2023] [Accepted: 07/21/2023] [Indexed: 08/22/2023] Open
Abstract
Mutations of the GABA-A receptor subunit β1 (GABRB1) gene are found in autism patients. However, it remains unclear how mutations in Gabrb1 may lead to autism. We generated Gabrb1-/- mouse model, which showed autistic-like behaviors. We carried out RNA-seq on the hippocampus and found glutamatergic pathway may be involved. We further carried out single-cell RNA sequencing on the whole brain followed by qRT-PCR, immunofluorescence, electrophysiology, and metabolite detection on specific cell types. We identified the up-regulated Glul/Slc38a3 in astrocytes, Grin1/Grin2b in neurons, glutamate, and the ratio of Glu/GABA in the hippocampus. Consistent with these results, increased NMDAR-currents and reduced GABAAR-currents in the CA1 neurons were detected in Gabrb1-/- mice. NMDAR antagonist memantine or Glul inhibitor methionine sulfoximine could rescue the abnormal behaviors in Gabrb1-/- mice. Our data reveal that upregulation of the glutamatergic synapse pathway, including NMDARs at neuronal synapses and glutamine exported by astrocytes, may lead to autistic-like behaviors.
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Affiliation(s)
- Jing Wang
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
| | - Yue Gao
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Guangdong Key Laboratory of Psychiatric Disorders, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Liuyan Xiao
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Guangdong Key Laboratory of Psychiatric Disorders, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Yanmei Lin
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Psychiatry, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
| | - Lang Huang
- Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Guangdong Key Laboratory of Psychiatric Disorders, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Jinfa Chen
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Psychiatry, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Guanmei Liang
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Weiming Li
- Department of Psychiatry, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
| | - Wenjuan Yi
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
| | - Jianpei Lao
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Psychiatry, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Bin Zhang
- Department of Psychiatry, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
| | - Tian-Ming Gao
- Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Guangdong Key Laboratory of Psychiatric Disorders, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Mei Zhong
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
| | - Xinping Yang
- Center for Genetics and Developmental Systems Biology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Psychiatry, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
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14
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Phan HTL, Kim K, Lee H, Seong JK. Progress in and Prospects of Genome Editing Tools for Human Disease Model Development and Therapeutic Applications. Genes (Basel) 2023; 14:483. [PMID: 36833410 PMCID: PMC9957140 DOI: 10.3390/genes14020483] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Revised: 02/09/2023] [Accepted: 02/10/2023] [Indexed: 02/17/2023] Open
Abstract
Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, are widely accepted because of their diversity and enormous potential for targeted genomic modifications in eukaryotes and other animals. Moreover, rapid advances in genome editing tools have accelerated the ability to produce various genetically modified animal models for studying human diseases. Given the advances in gene editing tools, these animal models are gradually evolving toward mimicking human diseases through the introduction of human pathogenic mutations in their genome rather than the conventional gene knockout. In the present review, we summarize the current progress in and discuss the prospects for developing mouse models of human diseases and their therapeutic applications based on advances in the study of programmable nucleases.
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Affiliation(s)
- Hong Thi Lam Phan
- Department of Physiology, Korea University College of Medicine, Seoul 02841, Republic of Korea
| | - Kyoungmi Kim
- Department of Physiology, Korea University College of Medicine, Seoul 02841, Republic of Korea
- Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea
| | - Ho Lee
- Graduate School of Cancer Science and Policy, National Cancer Center, Goyang 10408, Republic of Korea
| | - Je Kyung Seong
- Korea Mouse Phenotyping Center, Seoul National University, Seoul 08826, Republic of Korea
- Laboratory of Developmental Biology and Genomics, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea
- Interdisciplinary Program for Bioinformatics, Program for Cancer Biology, BIO-MAX/N-Bio Institute, Seoul National University, Seoul 08826, Republic of Korea
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15
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Goto T, Yogo K, Hochi S, Hirabayashi M. Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease. Mol Biol Rep 2023; 50:1231-1239. [PMID: 36441374 DOI: 10.1007/s11033-022-08054-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Accepted: 10/19/2022] [Indexed: 11/30/2022]
Abstract
BACKGROUND The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA. METHODS AND RESULTS We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60 ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7-20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60 ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction. CONCLUSIONS The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.
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Affiliation(s)
- Teppei Goto
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, 444-8787, Okazaki, Aichi, Japan.,Laboratory for Comparative Connectomics, RIKEN Center for Biosystems Dynamics Research, 650-0047, Kobe, Hyogo, Japan
| | - Kyoko Yogo
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, 444-8787, Okazaki, Aichi, Japan
| | - Shinichi Hochi
- Faculty of Textile Science and Technology, Shinshu University, 386-8567, Ueda, Nagano, Japan
| | - Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, 444-8787, Okazaki, Aichi, Japan. .,The Graduate University of Advanced Studies, 444-8787, Okazaki, Aichi, Japan.
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16
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Abstract
Many genome-edited mouse and rat strains have been produced using engineered endonucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Especially, CRISPR-Cas9 is powerful tool that can be easy, rapid, and high-efficiency-produced new genome-edited strains. Furthermore, new technique, Technique for Animal Knockout system by Electroporation (TAKE), efficiently accelerate production of new strains by direct nuclease introduction into intact embryos using electroporation. This chapter presents a latest technical information in the production of genome-edited mouse and rat by TAKE method.
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Affiliation(s)
- Takehito Kaneko
- Division of Fundamental and Applied Sciences, Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan.
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17
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Wani AK, Akhtar N, Singh R, Prakash A, Raza SHA, Cavalu S, Chopra C, Madkour M, Elolimy A, Hashem NM. Genome centric engineering using ZFNs, TALENs and CRISPR-Cas9 systems for trait improvement and disease control in Animals. Vet Res Commun 2023; 47:1-16. [PMID: 35781172 DOI: 10.1007/s11259-022-09967-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Accepted: 06/24/2022] [Indexed: 01/27/2023]
Abstract
Livestock is an essential life commodity in modern agriculture involving breeding and maintenance. The farming practices have evolved mainly over the last century for commercial outputs, animal welfare, environment friendliness, and public health. Modifying genetic makeup of livestock has been proposed as an effective tool to create farmed animals with characteristics meeting modern farming system goals. The first technique used to produce transgenic farmed animals resulted in random transgene insertion and a low gene transfection rate. Therefore, genome manipulation technologies have been developed to enable efficient gene targeting with a higher accuracy and gene stability. Genome editing (GE) with engineered nucleases-Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) regulates the targeted genetic alterations to facilitate multiple genomic modifications through protein-DNA binding. The application of genome editors indicates usefulness in reproduction, animal models, transgenic animals, and cell lines. Recently, CRISPR/Cas system, an RNA-dependent genome editing tool (GET), is considered one of the most advanced and precise GE techniques for on-target modifications in the mammalian genome by mediating knock-in (KI) and knock-out (KO) of several genes. Lately, CRISPR/Cas9 tool has become the method of choice for genome alterations in livestock species due to its efficiency and specificity. The aim of this review is to discuss the evolution of engineered nucleases and GETs as a powerful tool for genome manipulation with special emphasis on its applications in improving economic traits and conferring resistance to infectious diseases of animals used for food production, by highlighting the recent trends for maintaining sustainable livestock production.
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Affiliation(s)
- Atif Khurshid Wani
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Nahid Akhtar
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Reena Singh
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Ajit Prakash
- Department of Biochemistry and Biophysics, University of North Carolina, 120 Mason Farm Road, CB# 7260, 3093 Genetic Medicine, Chapel Hill, NC, 27599-2760, USA
| | - Sayed Haidar Abbas Raza
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Simona Cavalu
- Faculty of Medicine and Pharmacy, University of Oradea, P -ta 1Decembrie 10, 410073, Oradea, Romania
| | - Chirag Chopra
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Mahmoud Madkour
- Animal Production Department, National Research Centre, Dokki, Giza, 12622, Egypt
| | - Ahmed Elolimy
- Animal Production Department, National Research Centre, Dokki, Giza, 12622, Egypt
| | - Nesrein M Hashem
- Department of Animal and Fish Production, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, 21545, Egypt.
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18
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Ozawa M, Taguchi J, Katsuma K, Ishikawa-Yamauchi Y, Kikuchi M, Sakamoto R, Yamada Y, Ikawa M. Efficient simultaneous double DNA knock-in in murine embryonic stem cells by CRISPR/Cas9 ribonucleoprotein-mediated circular plasmid targeting for generating gene-manipulated mice. Sci Rep 2022; 12:21558. [PMID: 36513736 PMCID: PMC9748034 DOI: 10.1038/s41598-022-26107-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Accepted: 12/09/2022] [Indexed: 12/15/2022] Open
Abstract
Gene targeting of embryonic stem (ES) cells followed by chimera production has been conventionally used for developing gene-manipulated mice. Although direct knock-in (KI) using murine zygote via CRISPR/Cas9-mediated genome editing has been reported, ES cell targeting still has merits, e.g., high throughput work can be performed in vitro. In this study, we first compared the KI efficiency of mouse ES cells with CRISPR/Cas9 expression vector and ribonucleoprotein (RNP), and confirmed that KI efficiency was significantly increased by using RNP. Using CRISPR/Cas9 RNP and circular plasmid with homologous arms as a targeting vector, knock-in within ES cell clones could be obtained efficiently without drug selection, thus potentially shortening the vector construction or cell culture period. Moreover, by incorporating a drug-resistant cassette into the targeting vectors, double DNA KI can be simultaneously achieved at high efficiency by a single electroporation. This technique will help to facilitate the production of genetically modified mouse models that are fundamental for exploring topics related to human and mammalian biology.
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Affiliation(s)
- Manabu Ozawa
- grid.26999.3d0000 0001 2151 536XLaboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Jumpei Taguchi
- grid.26999.3d0000 0001 2151 536XDivision of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Kento Katsuma
- grid.26999.3d0000 0001 2151 536XLaboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Yu Ishikawa-Yamauchi
- grid.26999.3d0000 0001 2151 536XLaboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Mio Kikuchi
- grid.26999.3d0000 0001 2151 536XDivision of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Reiko Sakamoto
- grid.26999.3d0000 0001 2151 536XDivision of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Yasuhiro Yamada
- grid.26999.3d0000 0001 2151 536XDivision of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Masahito Ikawa
- grid.26999.3d0000 0001 2151 536XLaboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan ,grid.136593.b0000 0004 0373 3971Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871 Japan
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19
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Schweizer U, Fabiano M. Selenoproteins in brain development and function. Free Radic Biol Med 2022; 190:105-115. [PMID: 35961466 DOI: 10.1016/j.freeradbiomed.2022.07.022] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Revised: 06/21/2022] [Accepted: 07/26/2022] [Indexed: 01/18/2023]
Abstract
Expression of selenoproteins is widespread in neurons of the central nervous system. There is continuous evidence presented over decades that low levels of selenium or selenoproteins are linked to seizures and epilepsy indicating a failure of the inhibitory system. Many developmental processes in the brain depend on the thyroid hormone T3. T3 levels can be locally increased by the action of iodothyronine deiodinases on the prohormone T4. Since deiodinases are selenoproteins, it is expected that selenoprotein deficiency may affect development of the central nervous system. Studies in genetically modified mice or clinical observations of patients with rare diseases point to a role of selenoproteins in brain development and degeneration. In particular selenoprotein P is central to brain function by virtue of its selenium transport function into and within the brain. We summarize which selenoproteins are essential for the brain, which processes depend on selenoproteins, and what is known about genetic deficiencies of selenoproteins in humans. This review is not intended to cover the potential influence of selenium or selenoproteins on major neurodegenerative disorders in human.
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Affiliation(s)
- Ulrich Schweizer
- Institut für Biochemie und Molekularbiologie, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, Nussallee 11, 53115, Bonn, Germany.
| | - Marietta Fabiano
- Institut für Biochemie und Molekularbiologie, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, Nussallee 11, 53115, Bonn, Germany
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20
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Hawsawi YM, Shams A, Theyab A, Siddiqui J, Barnawee M, Abdali WA, Marghalani NA, Alshelali NH, Al-Sayed R, Alzahrani O, Alqahtani A, Alsulaiman AM. The State-of-the-Art of Gene Editing and its Application to Viral Infections and Diseases Including COVID-19. Front Cell Infect Microbiol 2022; 12:869889. [PMID: 35782122 PMCID: PMC9241565 DOI: 10.3389/fcimb.2022.869889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2022] [Accepted: 05/09/2022] [Indexed: 11/26/2022] Open
Abstract
Gene therapy delivers a promising hope to cure many diseases and defects. The discovery of gene-editing technology fueled the world with valuable tools that have been employed in various domains of science, medicine, and biotechnology. Multiple means of gene editing have been established, including CRISPR/Cas, ZFNs, and TALENs. These strategies are believed to help understand the biological mechanisms of disease progression. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been designated the causative virus for coronavirus disease 2019 (COVID-19) that emerged at the end of 2019. This viral infection is a highly pathogenic and transmissible disease that caused a public health pandemic. As gene editing tools have shown great success in multiple scientific and medical areas, they could eventually contribute to discovering novel therapeutic and diagnostic strategies to battle the COVID-19 pandemic disease. This review aims to briefly highlight the history and some of the recent advancements of gene editing technologies. After that, we will describe various biological features of the CRISPR-Cas9 system and its diverse implications in treating different infectious diseases, both viral and non-viral. Finally, we will present current and future advancements in combating COVID-19 with a potential contribution of the CRISPR system as an antiviral modality in this battle.
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Affiliation(s)
- Yousef M. Hawsawi
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
- College of Medicine, Al-Faisal University, Riyadh, Saudi Arabia
| | - Anwar Shams
- Department of Pharmacology, College of Medicine, Taif University, Mecca, Saudi Arabia
| | - Abdulrahman Theyab
- College of Medicine, Al-Faisal University, Riyadh, Saudi Arabia
- Department of Laboratory & Blood Bank, Security Forces Hospital, Mecca, Saudi Arabia
| | - Jumana Siddiqui
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Mawada Barnawee
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Wed A. Abdali
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Nada A. Marghalani
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Nada H. Alshelali
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Rawan Al-Sayed
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Othman Alzahrani
- Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Saudi Arabia
- Genome and Biotechnology Unit, Faculty of Science, University of Tabuk, Tabuk, Saudi Arabia
| | - Alanoud Alqahtani
- Bristol Medical School, Faculty of Health Sciences, University of Bristol, Bristol, United Kingdom
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21
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3R measures in facilities for the production of genetically modified rodents. Lab Anim (NY) 2022; 51:162-177. [PMID: 35641635 DOI: 10.1038/s41684-022-00978-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2021] [Accepted: 04/22/2022] [Indexed: 12/30/2022]
Abstract
Sociocultural changes in the human-animal relationship have led to increasing demands for animal welfare in biomedical research. The 3R concept is the basis for bringing this demand into practice: Replace animal experiments with alternatives where possible, Reduce the number of animals used to a scientifically justified minimum and Refine the procedure to minimize animal harm. The generation of gene-modified sentient animals such as mice and rats involves many steps that include various forms of manipulation. So far, no coherent analysis of the application of the 3Rs to gene manipulation has been performed. Here we provide guidelines from the Committee on Genetics and Breeding of Laboratory Animals of the German Society for Laboratory Animal Science to implement the 3Rs in every step during the generation of genetically modified animals. We provide recommendations for applying the 3Rs as well as success/intervention parameters for each step of the process, from experiment planning to choice of technology, harm-benefit analysis, husbandry conditions, management of genetically modified lines and actual procedures. We also discuss future challenges for animal welfare in the context of developing technologies. Taken together, we expect that our comprehensive analysis and our recommendations for the appropriate implementation of the 3Rs to technologies for genetic modifications of rodents will benefit scientists from a wide range of disciplines and will help to improve the welfare of a large number of laboratory animals worldwide.
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22
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Abstract
The salamander Ambystoma mexicanum, commonly called "the axolotl" has a long, illustrious history as a model organism, perhaps with one of the longest track records as a laboratory-bred vertebrate, yet it also holds a prominent place among the emerging model organisms. Or rather it is by now an "emerged" model organism, boasting a full cohort molecular genetic tools that allows an expanding community of researchers in the field to explore the remarkable traits of this animal including regeneration, at cellular and molecular precision-which had been a dream for researchers over the years. This chapter describes the journey to this status, that could be helpful for those developing their respective animal or plant models.
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Affiliation(s)
- Karen Echeverri
- Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA, United States
| | - Jifeng Fei
- Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China
| | - Elly M Tanaka
- Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.
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23
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Hong CH, Ko MS, Kim JH, Cho H, Lee CH, Yoon JE, Yun JY, Baek IJ, Jang JE, Lee SE, Cho YK, Baek JY, Oh SJ, Lee BY, Lim JS, Lee J, Hartig SM, Conde de la Rosa L, Garcia-Ruiz C, Lee KU, Fernández-Checa JC, Choi JW, Kim S, Koh EH. Sphingosine 1-Phosphate Receptor 4 Promotes Nonalcoholic Steatohepatitis by Activating NLRP3 Inflammasome. Cell Mol Gastroenterol Hepatol 2021; 13:925-947. [PMID: 34890841 PMCID: PMC8810559 DOI: 10.1016/j.jcmgh.2021.12.002] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/07/2021] [Revised: 12/02/2021] [Accepted: 12/02/2021] [Indexed: 12/12/2022]
Abstract
BACKGROUND & AIMS Sphingosine 1-phosphate receptors (S1PRs) are a group of G-protein-coupled receptors that confer a broad range of functional effects in chronic inflammatory and metabolic diseases. S1PRs also may mediate the development of nonalcoholic steatohepatitis (NASH), but the specific subtypes involved and the mechanism of action are unclear. METHODS We investigated which type of S1PR isoforms is activated in various murine models of NASH. The mechanism of action of S1PR4 was examined in hepatic macrophages isolated from high-fat, high-cholesterol diet (HFHCD)-fed mice. We developed a selective S1PR4 functional antagonist by screening the fingolimod (2-amino-2-[2-(4- n -octylphenyl)ethyl]-1,3- propanediol hydrochloride)-like sphingolipid-focused library. RESULTS The livers of various mouse models of NASH as well as hepatic macrophages showed high expression of S1pr4. Moreover, in a cohort of NASH patients, expression of S1PR4 was 6-fold higher than those of healthy controls. S1pr4+/- mice were protected from HFHCD-induced NASH and hepatic fibrosis without changes in steatosis. S1pr4 depletion in hepatic macrophages inhibited lipopolysaccharide-mediated Ca++ release and deactivated the Nod-like receptor pyrin domain-containning protein 3 (NLRP3) inflammasome. S1P increased the expression of S1pr4 in hepatic macrophages and activated NLRP3 inflammasome through inositol trisphosphate/inositol trisphosphate-receptor-dependent [Ca++] signaling. To further clarify the biological function of S1PR4, we developed SLB736, a novel selective functional antagonist of SIPR4. Similar to S1pr4+/- mice, administration of SLB736 to HFHCD-fed mice prevented the development of NASH and hepatic fibrosis, but not steatosis, by deactivating the NLRP3 inflammasome. CONCLUSIONS S1PR4 may be a new therapeutic target for NASH that mediates the activation of NLRP3 inflammasome in hepatic macrophages.
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Affiliation(s)
- Chung Hwan Hong
- Department of Medical Science, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Myoung Seok Ko
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Jae Hyun Kim
- College of Pharmacy, Seoul National University, Seoul, Korea,College of Pharmacy, Kangwon National University, Chuncheon, Korea
| | - Hyunkyung Cho
- College of Pharmacy, Seoul National University, Seoul, Korea
| | - Chi-Ho Lee
- College of Pharmacy, Gachon University, Incheon, Korea
| | - Ji Eun Yoon
- Department of Medical Science, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Ji-Young Yun
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - In-Jeoung Baek
- Convergence Medicine Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea,Department of Convergence Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Jung Eun Jang
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Seung Eun Lee
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Yun Kyung Cho
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Ji Yeon Baek
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Soo Jin Oh
- New Drug Development Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | | | - Joon Seo Lim
- Clinical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Jongkook Lee
- College of Pharmacy, Kangwon National University, Chuncheon, Korea
| | - Sean M. Hartig
- Molecular and Cellular Biology, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, Texas
| | - Laura Conde de la Rosa
- Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona and Liver Unit-Hospital Clinic-Instituto de Investigaciones Biomédicas August Pi i Sunyer, Centro de Investigación Biomédica en Red, Barcelona, Spain
| | - Carmen Garcia-Ruiz
- Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona and Liver Unit-Hospital Clinic-Instituto de Investigaciones Biomédicas August Pi i Sunyer, Centro de Investigación Biomédica en Red, Barcelona, Spain,Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Ki-Up Lee
- Department of Convergence Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Jose C. Fernández-Checa
- Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona and Liver Unit-Hospital Clinic-Instituto de Investigaciones Biomédicas August Pi i Sunyer, Centro de Investigación Biomédica en Red, Barcelona, Spain,Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, California,Correspondence Address correspondence to: Jose C. Fernández-Checa, PhD, Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Cientificas, Barcelona and Liver Unit-Hospital Clinic–Instituto de Investigaciones Biomédicas August Pi i Sunyer, Centro de Investigación Biomédica en Red, Barcelona 08036, Spain. fax: (34) 93-3129405.
| | - Ji Woong Choi
- College of Pharmacy, Gachon University, Incheon, Korea,Ji Woong Choi, PhD, Laboratory of Pharmacology, College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon 21936, Korea. fax: (82) 32-820-4829.
| | - Sanghee Kim
- College of Pharmacy, Seoul National University, Seoul, Korea,Sanghee Kim, PhD, College of Pharmacy, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul 08826, Korea. fax: (82) 2-762-8322.
| | - Eun Hee Koh
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea,Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea,Eun Hee Koh, MD, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea. fax: (82) 2-3010-6962.
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24
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Dehdilani N, Taemeh SY, Goshayeshi L, Dehghani H. Genetically engineered birds; pre-CRISPR and CRISPR era. Biol Reprod 2021; 106:24-46. [PMID: 34668968 DOI: 10.1093/biolre/ioab196] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 10/08/2021] [Accepted: 10/14/2021] [Indexed: 11/14/2022] Open
Abstract
Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-CRISPR and CRISPR era in the field of avian genome engineering.
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Affiliation(s)
- Nima Dehdilani
- Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Sara Yousefi Taemeh
- Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Lena Goshayeshi
- Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Hesam Dehghani
- Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.,Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.,Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
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25
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Koh EH, Yoon JE, Ko MS, Leem J, Yun JY, Hong CH, Cho YK, Lee SE, Jang JE, Baek JY, Yoo HJ, Kim SJ, Sung CO, Lim JS, Jeong WI, Back SH, Baek IJ, Torres S, Solsona-Vilarrasa E, Conde de la Rosa L, Garcia-Ruiz C, Feldstein AE, Fernandez-Checa JC, Lee KU. Sphingomyelin synthase 1 mediates hepatocyte pyroptosis to trigger non-alcoholic steatohepatitis. Gut 2021; 70:1954-1964. [PMID: 33208407 PMCID: PMC8458090 DOI: 10.1136/gutjnl-2020-322509] [Citation(s) in RCA: 110] [Impact Index Per Article: 27.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Revised: 10/26/2020] [Accepted: 10/26/2020] [Indexed: 02/06/2023]
Abstract
OBJECTIVE Lipotoxic hepatocyte injury is a primary event in non-alcoholic steatohepatitis (NASH), but the mechanisms of lipotoxicity are not fully defined. Sphingolipids and free cholesterol (FC) mediate hepatocyte injury, but their link in NASH has not been explored. We examined the role of free cholesterol and sphingomyelin synthases (SMSs) that generate sphingomyelin (SM) and diacylglycerol (DAG) in hepatocyte pyroptosis, a specific form of programmed cell death associated with inflammasome activation, and NASH. DESIGN Wild-type C57BL/6J mice were fed a high fat and high cholesterol diet (HFHCD) to induce NASH. Hepatic SMS1 and SMS2 expressions were examined in various mouse models including HFHCD-fed mice and patients with NASH. Pyroptosis was estimated by the generation of the gasdermin-D N-terminal fragment. NASH susceptibility and pyroptosis were examined following knockdown of SMS1, protein kinase Cδ (PKCδ), or the NLR family CARD domain-containing protein 4 (NLRC4). RESULTS HFHCD increased the hepatic levels of SM and DAG while decreasing the level of phosphatidylcholine. Hepatic expression of Sms1 but not Sms2 was higher in mouse models and patients with NASH. FC in hepatocytes induced Sms1 expression, and Sms1 knockdown prevented HFHCD-induced NASH. DAG produced by SMS1 activated PKCδ and NLRC4 inflammasome to induce hepatocyte pyroptosis. Depletion of Nlrc4 prevented hepatocyte pyroptosis and the development of NASH. Conditioned media from pyroptotic hepatocytes activated the NOD-like receptor family pyrin domain containing 3 inflammasome (NLRP3) in Kupffer cells, but Nlrp3 knockout mice were not protected against HFHCD-induced hepatocyte pyroptosis. CONCLUSION SMS1 mediates hepatocyte pyroptosis through a novel DAG-PKCδ-NLRC4 axis and holds promise as a therapeutic target for NASH.
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Affiliation(s)
- Eun Hee Koh
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Ji Eun Yoon
- Department of Medical Science, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Myoung Seok Ko
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Jaechan Leem
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Ji-Young Yun
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Chung Hwan Hong
- Department of Medical Science, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Yun Kyung Cho
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Seung Eun Lee
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Jung Eun Jang
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Ji Yeon Baek
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Hyun Ju Yoo
- The Convergence Medicine Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Su Jung Kim
- The Convergence Medicine Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Chang Ohk Sung
- Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Joon Seo Lim
- Clinical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Won-Il Jeong
- Laboratory of Liver Research, Graduate School of Medical Science and Engineering, KAIST, Daejeon, South Korea
| | - Sung Hoon Back
- School of Biological Sciences, University of Ulsan, Ulsan, South Korea
| | - In-Jeoung Baek
- The Convergence Medicine Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
| | - Sandra Torres
- Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona (IIBB), CSIC, Barcelona, Spain and Liver Unit-IDIBAPS and Centro de Investigación Biomédica en Red (CIBERehd), Barcelona, Spain
| | - Estel Solsona-Vilarrasa
- Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona (IIBB), CSIC, Barcelona, Spain and Liver Unit-IDIBAPS and Centro de Investigación Biomédica en Red (CIBERehd), Barcelona, Spain
| | - Laura Conde de la Rosa
- Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona (IIBB), CSIC, Barcelona, Spain and Liver Unit-IDIBAPS and Centro de Investigación Biomédica en Red (CIBERehd), Barcelona, Spain
| | - Carmen Garcia-Ruiz
- Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona (IIBB), CSIC, Barcelona, Spain and Liver Unit-IDIBAPS and Centro de Investigación Biomédica en Red (CIBERehd), Barcelona, Spain,University of Southern California Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Ariel E Feldstein
- Department of Pediatrics, University of California San Diego, La Jolla, CA, USA
| | - Jose C Fernandez-Checa
- Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona (IIBB), CSIC, Barcelona, Spain and Liver Unit-IDIBAPS and Centro de Investigación Biomédica en Red (CIBERehd), Barcelona, Spain .,University of Southern California Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Ki-Up Lee
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
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26
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Shih PY, Hsieh BY, Lin MH, Huang TN, Tsai CY, Pong WL, Lee SP, Hsueh YP. CTTNBP2 Controls Synaptic Expression of Zinc-Related Autism-Associated Proteins and Regulates Synapse Formation and Autism-like Behaviors. Cell Rep 2021; 31:107700. [PMID: 32492416 DOI: 10.1016/j.celrep.2020.107700] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2019] [Revised: 03/29/2020] [Accepted: 05/06/2020] [Indexed: 12/12/2022] Open
Abstract
Synaptic dysregulation is a critical feature of autism spectrum disorders (ASDs). Among various autism-associated genes, cortactin binding protein 2 (CTTNBP2) is a cytoskeleton regulator predominantly expressed in neurons and highly enriched at dendritic spines. Here, using Cttnbp2 knockout and ASD-linked mutant mice, we demonstrate that Cttnbp2 deficiency reduces zinc levels in the brain, alters synaptic protein targeting, impairs dendritic spine formation and ultrastructure of postsynaptic density, and influences neuronal activation and autism-like behaviors. A link to autism, the NMDAR-SHANK pathway, and zinc-related regulation are three features shared by CTTNBP2-regulated synaptic proteins. Zinc supplementation rescues the synaptic expression of CTTNBP2-regulated proteins. Moreover, zinc supplementation and administration of D-cycloserine, an NMDAR coagonist, improve the social behaviors of Cttnbp2-deficient mice. We suggest that CTTNBP2 controls the synaptic expression of a set of zinc-regulated autism-associated genes and influences NMDAR function and signaling, providing an example of how genetic and environmental factor crosstalk controls social behaviors.
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Affiliation(s)
- Pu-Yun Shih
- Molecular and Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China; Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
| | - Bing-Yuan Hsieh
- Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
| | - Ming-Hui Lin
- Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
| | - Tzyy-Nan Huang
- Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
| | - Ching-Yen Tsai
- Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
| | - Wen-Li Pong
- Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
| | - Sue-Ping Lee
- Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
| | - Yi-Ping Hsueh
- Molecular and Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China; Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China.
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27
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Allen KP, Csida TJ, Thulin JD. Assessing Accumulation of Organic Material on Rodent Cage Accessories. JOURNAL OF THE AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE 2021; 60:281-288. [PMID: 33673882 DOI: 10.30802/aalas-jaalas-20-000087] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
According to the 8th edition of the Guide for the Care and Use of Laboratory Animals (the Guide), rodent cage accessories, such as filter tops, should be sanitized at least once every 2 wk. We performed a study to test the hypothesis that organic contamination (measured by ATP content, expressed as relative light units (RLU)) of cage accessories (wire bar inserts and filter top lids) does not differ at 2 wk (14 d) as compared with 30, 60, and 90-d time points after cage change even when in constant use. An additional time point for filter top lids of 180 d after cage change was also evaluated. Eight groups were studied: the wire bar inserts and filter top lids used for mice and rats, in both static and individually ventilated cages (IVC). When analyzing data from both mouse and rat static and IVC caging, we found that the mean RLU values for mouse IVC and rat static and IVC cage components were below 100,000 RLU at the 14-d time point. The mean value for the mouse static group was slightly above 100,000 RLU at this time point. Based on this observation, we considered 100,000 RLU to be an appropriate actionable level. We concluded that changing wire bar inserts at least every 14 d, as recommended in the Guide for sanitizing these components in mouse and rat static cages, may be considered acceptable. This interval could be extended for mouse and rat IVC cages up to 90 d while remaining below this limit. Filter top lids for mouse static cages should be changed at least every 30 d, but static rat and IVC mouse/rat filter top lids could be changed up to every 180 d, while still staying below this actionable level of contamination.
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Affiliation(s)
- Kenneth P Allen
- Biomedical Resource Center, Office of Research, Milwaukee, Wisconsin; Department of Microbiology and Molecular Genetics, Milwaukee, Wisconsin;,
| | - Tarrant J Csida
- Biomedical Resource Center, Office of Research, Milwaukee, Wisconsin
| | - Joseph D Thulin
- Biomedical Resource Center, Office of Research, Milwaukee, Wisconsin; Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin
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28
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Ko MS, Yun JY, Baek IJ, Jang JE, Hwang JJ, Lee SE, Heo SH, Bader DA, Lee CH, Han J, Moon JS, Lee JM, Hong EG, Lee IK, Kim SW, Park JY, Hartig SM, Kang UJ, Moore DD, Koh EH, Lee KU. Mitophagy deficiency increases NLRP3 to induce brown fat dysfunction in mice. Autophagy 2021; 17:1205-1221. [PMID: 32400277 PMCID: PMC8143238 DOI: 10.1080/15548627.2020.1753002] [Citation(s) in RCA: 64] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 03/24/2020] [Accepted: 04/03/2020] [Indexed: 12/22/2022] Open
Abstract
Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases.Abbreviations: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.
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Affiliation(s)
- Myoung Seok Ko
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Ji Young Yun
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - In-Jeoung Baek
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Jung Eun Jang
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Jung Jin Hwang
- Institute for Innovative Cancer Research, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Seung Eun Lee
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Seung-Ho Heo
- Convergence Medicine Research Center, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - David A. Bader
- Molecular and Cellular Biology and Medicine, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, Texas, USA
| | - Chul-Ho Lee
- Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
| | - Jaeseok Han
- Soonchunhyang Institute of Med-bio Science (SIMS), Soonchunhyang University, Korea
| | - Jong-Seok Moon
- Soonchunhyang Institute of Med-bio Science (SIMS), Soonchunhyang University, Korea
| | - Jae Man Lee
- Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu, Korea
| | - Eun-Gyoung Hong
- Division of Endocrinology and Metabolism, Department of Internal Medicine, Hallym University Dongtan Sacred Heart Hospital, Hallym University College of Medicine, Hwaseong, Korea
| | - In-Kyu Lee
- Department of Internal Medicine and Biochemistry, Kyungpook National University School of Medicine, Daegu, Korea
| | - Seong Who Kim
- Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, Korea
| | - Joong Yeol Park
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Sean M. Hartig
- Molecular and Cellular Biology and Medicine, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, Texas, USA
| | - Un Jung Kang
- Department of Neurology, Neuroscience and Physiology, Marlene and Paolo Fresco Institute for Parkinson’s and Movement Disorders, NYU Langone Health, New York, USA
| | - David D. Moore
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
| | - Eun Hee Koh
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Ki-up Lee
- Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
- Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
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29
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Chenouard V, Remy S, Tesson L, Ménoret S, Ouisse LH, Cherifi Y, Anegon I. Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models. Front Genet 2021; 12:615491. [PMID: 33959146 PMCID: PMC8093876 DOI: 10.3389/fgene.2021.615491] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 02/22/2021] [Indexed: 12/13/2022] Open
Abstract
The rat has been extensively used as a small animal model. Many genetically engineered rat models have emerged in the last two decades, and the advent of gene-specific nucleases has accelerated their generation in recent years. This review covers the techniques and advances used to generate genetically engineered rat lines and their application to the development of rat models more broadly, such as conditional knockouts and reporter gene strains. In addition, genome-editing techniques that remain to be explored in the rat are discussed. The review also focuses more particularly on two areas in which extensive work has been done: human genetic diseases and immune system analysis. Models are thoroughly described in these two areas and highlight the competitive advantages of rat models over available corresponding mouse versions. The objective of this review is to provide a comprehensive description of the advantages and potential of rat models for addressing specific scientific questions and to characterize the best genome-engineering tools for developing new projects.
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Affiliation(s)
- Vanessa Chenouard
- CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Université de Nantes, Nantes, France
- genOway, Lyon, France
| | - Séverine Remy
- CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Université de Nantes, Nantes, France
| | - Laurent Tesson
- CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Université de Nantes, Nantes, France
| | - Séverine Ménoret
- CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Université de Nantes, Nantes, France
- CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016, CNRS UMS 3556, Nantes Université, Nantes, France
| | - Laure-Hélène Ouisse
- CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Université de Nantes, Nantes, France
| | | | - Ignacio Anegon
- CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Université de Nantes, Nantes, France
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30
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Kim H, Lee K, Kim JM, Kim MY, Kim JR, Lee HW, Chung YW, Shin HI, Kim T, Park ES, Rho J, Lee SH, Kim N, Lee SY, Choi Y, Jeong D. Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts. Nat Commun 2021; 12:2258. [PMID: 33859201 PMCID: PMC8050258 DOI: 10.1038/s41467-021-22565-7] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2018] [Accepted: 03/18/2021] [Indexed: 11/09/2022] Open
Abstract
Selenoproteins containing selenium in the form of selenocysteine are critical for bone remodeling. However, their underlying mechanism of action is not fully understood. Herein, we report the identification of selenoprotein W (SELENOW) through large-scale mRNA profiling of receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation, as a protein that is downregulated via RANKL/RANK/tumour necrosis factor receptor-associated factor 6/p38 signaling. RNA-sequencing analysis revealed that SELENOW regulates osteoclastogenic genes. SELENOW overexpression enhances osteoclastogenesis in vitro via nuclear translocation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 mediated by 14-3-3γ, whereas its deficiency suppresses osteoclast formation. SELENOW-deficient and SELENOW-overexpressing mice exhibit high bone mass phenotype and osteoporosis, respectively. Ectopic SELENOW expression stimulates cell-cell fusion critical for osteoclast maturation as well as bone resorption. Thus, RANKL-dependent repression of SELENOW regulates osteoclast differentiation and blocks osteoporosis caused by overactive osteoclasts. These findings demonstrate a biological link between selenium and bone metabolism. Selenoproteins containing selenium have a variety of physiological functions including redox homeostasis and thyroid hormone metabolism. Here, the authors show that RANKL-dependent repression of selenoprotein W regulates cell fusion during osteoclast differentiation and bone remodelling in mice.
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Affiliation(s)
- Hyunsoo Kim
- Department of Microbiology, Laboratory of Bone Metabolism and Control, Yeungnam University College of Medicine, Daegu, Korea.,Departments of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
| | - Kyunghee Lee
- Department of Microbiology, Laboratory of Bone Metabolism and Control, Yeungnam University College of Medicine, Daegu, Korea
| | - Jin Man Kim
- Department of Microbiology, Laboratory of Bone Metabolism and Control, Yeungnam University College of Medicine, Daegu, Korea
| | - Mi Yeong Kim
- Department of Microbiology, Laboratory of Bone Metabolism and Control, Yeungnam University College of Medicine, Daegu, Korea
| | - Jae-Ryong Kim
- Department of Biochemistry and Molecular Biology, Smart-aging Convergence Research Center, Yeungnam University College of Medicine, Daegu, Korea
| | - Han-Woong Lee
- Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea
| | - Youn Wook Chung
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea
| | - Hong-In Shin
- IHBR, Department of Oral Pathology, School of Dentistry, Kyungpook National University, Daegu, Korea
| | - Taesoo Kim
- Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon, Korea
| | - Eui-Soon Park
- Department of Microbiology and BK21 Bio Brain Center, Chungnam National University, Daejeon, Korea
| | - Jaerang Rho
- Department of Microbiology and BK21 Bio Brain Center, Chungnam National University, Daejeon, Korea
| | - Seoung Hoon Lee
- Department of Oral Microbiology and Immunology, Wonkwang University School of Dentistry, Iksan, Korea
| | - Nacksung Kim
- National Research Laboratory for Regulation of Bone Metabolism and Disease, Chonnam National University Medical School, Gwangju, Korea
| | - Soo Young Lee
- Division of Life and Pharmaceutical Sciences, Department of Life Science, Center for Cell Signaling & Drug Discovery Research, College of Natural Sciences, Ewha Womans University, Seoul, Korea
| | - Yongwon Choi
- Departments of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
| | - Daewon Jeong
- Department of Microbiology, Laboratory of Bone Metabolism and Control, Yeungnam University College of Medicine, Daegu, Korea.
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31
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The Genetic Basis of Reporter Mouse Strains. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021. [PMID: 33834450 DOI: 10.1007/978-981-33-6064-8_21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register]
Abstract
Genetically engineered mouse (GEM) models have been revolutionizing the biomedical studies on deciphering the physiological roles of genes in vivo. In addition to deactivating a gene in mice, diverse strategies have been created to monitor gene expressions and molecular dynamics of specific proteins in vivo. Although gene targeting in mouse embryonic stem (ES) cells was essential for the precise engineering of the mouse genome over almost three decades, this process is a time-consuming, expensive, and laborious one. These days, new technologies that directly apply engineered endonucleases, such as zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector (TALE) Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, into the mouse zygotes are enabling us to rapidly replace conventional gene targeting in mouse ES cells. In this chapter, we will describe the principles of reporter mouse strains and the recent advances in generating them using engineered endonucleases.
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32
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Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases. Nat Commun 2021; 12:1190. [PMID: 33608520 PMCID: PMC7895935 DOI: 10.1038/s41467-021-21464-1] [Citation(s) in RCA: 92] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Accepted: 01/28/2021] [Indexed: 02/07/2023] Open
Abstract
DddA-derived cytosine base editors (DdCBEs), composed of the split interbacterial toxin DddAtox, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), enable targeted C-to-T base conversions in mitochondrial DNA (mtDNA). Here, we demonstrate highly efficient mtDNA editing in mouse embryos using custom-designed DdCBEs. We target the mitochondrial gene, MT-ND5 (ND5), which encodes a subunit of NADH dehydrogenase that catalyzes NADH dehydration and electron transfer to ubiquinone, to obtain several mtDNA mutations, including m.G12918A associated with human mitochondrial diseases and m.C12336T that incorporates a premature stop codon, creating mitochondrial disease models in mice and demonstrating a potential for the treatment of mitochondrial disorders. Split DddA-derived base editors fused to TALEs enable mitochondrial DNA editing. Here the authors demonstrate their use in mouse embryos with germline transmission.
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33
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Tsunekawa Y, Terhune RK, Matsuzaki F. Protocol for De Novo Gene Targeting Via In Utero Electroporation. Methods Mol Biol 2021; 2312:309-320. [PMID: 34228299 DOI: 10.1007/978-1-0716-1441-9_19] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.
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Affiliation(s)
- Yuji Tsunekawa
- Laboratory for Cell Asymmetry, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
- Division of Molecular and Medical Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
| | | | - Fumio Matsuzaki
- Laboratory for Cell Asymmetry, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
- Laboratory of Molecular Cell Biology and Development, Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
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34
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Babochkina TI, Gerlinskaya LA, Moshkin MP. Generation of donor organs in chimeric animals via blastocyst complementation. Vavilovskii Zhurnal Genet Selektsii 2020; 24:913-921. [PMID: 35088005 PMCID: PMC8763716 DOI: 10.18699/vj20.690] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 10/21/2020] [Accepted: 11/17/2020] [Indexed: 11/25/2022] Open
Abstract
The lack of organs for transplantation is an important problem in medicine today. The growth of organs
in chimeric animals may be the solution of this. The proposed technology is the interspecific blastocyst complementation method in combination with genomic editing for obtaining “free niches” and pluripotent stem cell
production methods. The CRISPR/Cas9 method allows the so-called “free niches” to be obtained for blastocyst
complementation. The technologies of producing induced pluripotent stem cells give us the opportunity to obtain human donor cells capable of populating a “free niche”. Taken together, these technologies allow interspecific
blastocyst complementation between humans and other animals, which makes it possible in the future to grow
human organs for transplantations inside chimeric animals. However, in practice, in order to achieve successful
interspecific blastocyst complementation, it is necessary to solve a number of problems: to improve methods for
producing “chimeric competent” cells, to overcome specific interspecific barriers, to select compatible cell developmental stages for injection and the corresponding developmental stage of the host embryo, to prevent apoptosis of donor cells and to achieve effective proliferation of the human donor cells in the host animal. Also, it is
very important to analyze the ethical aspects related to developing technologies of chimeric organisms with the
participation of human cells. Today, many researchers are trying to solve these problems and also to establish new
approaches in the creation of interspecific chimeric organisms in order to grow human organs for transplantation.
In the present review we described the historical stages of the development of the blastocyst complementation
method, examined in detail the technologies that underlie modern blastocyst complementation, and analyzed
current progress that gives us the possibility to grow human organs in chimeric animals. We also considered the
barriers and issues preventing the successful implementation of interspecific blastocyst complementation in practice, and discussed the further development of this method
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Affiliation(s)
- T I Babochkina
- Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - L A Gerlinskaya
- Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - M P Moshkin
- Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
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35
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Abstract
The mouse is one of the most widely used model organisms for genetic study. The tools available to alter the mouse genome have developed over the preceding decades from forward screens to gene targeting in stem cells to the recent influx of CRISPR approaches. In this review, we first consider the history of mice in genetic study, the development of classic approaches to genome modification, and how such approaches have been used and improved in recent years. We then turn to the recent surge of nuclease-mediated techniques and how they are changing the field of mouse genetics. Finally, we survey common classes of alleles used in mice and discuss how they might be engineered using different methods.
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Affiliation(s)
- James F Clark
- Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mt. Sinai, New York, New York 10029, USA
| | - Colin J Dinsmore
- Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mt. Sinai, New York, New York 10029, USA
| | - Philippe Soriano
- Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mt. Sinai, New York, New York 10029, USA
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36
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Seita Y, Tsukiyama T, Azami T, Kobayashi K, Iwatani C, Tsuchiya H, Nakaya M, Tanabe H, Hitoshi S, Miyoshi H, Nakamura S, Kawauchi A, Ema M. Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†. Biol Reprod 2020; 100:1440-1452. [PMID: 30869744 DOI: 10.1093/biolre/ioz040] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2018] [Revised: 02/21/2019] [Accepted: 03/12/2019] [Indexed: 12/11/2022] Open
Abstract
Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.
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Affiliation(s)
- Yasunari Seita
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Tomoyuki Tsukiyama
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan.,Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Takuya Azami
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Kenichi Kobayashi
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan.,Department of Urology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Chizuru Iwatani
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Hideaki Tsuchiya
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Masataka Nakaya
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Hideyuki Tanabe
- Department of Evolutionary Studies of Biosystems, School of Advanced Sciences, SOKENDAI (The Graduate University for Advanced Studies), Hayama, Kanagawa, Japan
| | - Seiji Hitoshi
- Department of Physiology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Hiroyuki Miyoshi
- Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
| | - Shinichiro Nakamura
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Akihiro Kawauchi
- Department of Urology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Masatsugu Ema
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga, Japan.,Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Sakyo-ku, Kyoto, Japan.,PRESTO, Japan Science and Technology Agency, Honcho, Saitama, Japan
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37
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Blood disease-causing and -suppressing transcriptional enhancers: general principles and GATA2 mechanisms. Blood Adv 2020; 3:2045-2056. [PMID: 31289032 DOI: 10.1182/bloodadvances.2019000378] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2019] [Accepted: 05/29/2019] [Indexed: 12/16/2022] Open
Abstract
Intensive scrutiny of human genomes has unveiled considerable genetic variation in coding and noncoding regions. In cancers, including those of the hematopoietic system, genomic instability amplifies the complexity and functional consequences of variation. Although elucidating how variation impacts the protein-coding sequence is highly tractable, deciphering the functional consequences of variation in noncoding regions (genome reading), including potential transcriptional-regulatory sequences, remains challenging. A crux of this problem is the sheer abundance of gene-regulatory sequence motifs (cis elements) mediating protein-DNA interactions that are intermixed in the genome with thousands of look-alike sequences lacking the capacity to mediate functional interactions with proteins in vivo. Furthermore, transcriptional enhancers harbor clustered cis elements, and how altering a single cis element within a cluster impacts enhancer function is unpredictable. Strategies to discover functional enhancers have been innovated, and human genetics can provide vital clues to achieve this goal. Germline or acquired mutations in functionally critical (essential) enhancers, for example at the GATA2 locus encoding a master regulator of hematopoiesis, have been linked to human pathologies. Given the human interindividual genetic variation and complex genetic landscapes of hematologic malignancies, enhancer corruption, creation, and expropriation by new genes may not be exceedingly rare mechanisms underlying disease predisposition and etiology. Paradigms arising from dissecting essential enhancer mechanisms can guide genome-reading strategies to advance fundamental knowledge and precision medicine applications. In this review, we provide our perspective of general principles governing the function of blood disease-linked enhancers and GATA2-centric mechanisms.
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Kaneko T, Tanaka S. Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts. Biochem Biophys Res Commun 2020; 527:1039-1042. [PMID: 32439162 DOI: 10.1016/j.bbrc.2020.05.034] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Accepted: 05/06/2020] [Indexed: 11/28/2022]
Abstract
Many genome-edited animals have been produced using the CRISPR/Cas system. Genome-edited strains were produced by introducing nucleases into pronuclear stage embryos. Recently, a new electroporation technique (TAKE: Technique for Animal Knockout system by Electroporation) was developed for the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. Furthermore, this method, which can introduce nucleases into intact embryos, enables genome editing of mouse embryos in the oviducts. However, the present protocol required improvements for low litter size and restriction of operation time. In this study, the influence on the development and genome editing of mouse embryos in the oviducts by electroporation and operation time was examined. The genome-editing rate was higher in the embryos electroporated at 16:00-17:00 (PM) (54%) on the following day of natural mating compared to that of embryos at 10:00-11:00 (AM) (27%). The embryos at AM formed a complex with cumulus cells, and cumulus cells were freed from embryos by treatment with hyalronidase before electroporation. The results showed that the genome-editing rate was significantly increased in the embryos treated with hyalronidase at AM, because the cumulus cells surrounding the embryos interfered with the introduction of nucleases into embryos. This study demonstrated that it was possible to adjust the operation time for the introduction of nucleases into embryos in the oviducts by treatment with hyalronidase before electroporation. However, litter size and development of embryos after electroporation was quite low in all experiments (5-7) compared with the control without operation (11).
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Affiliation(s)
- Takehito Kaneko
- Division of Science and Engineering, Graduate School of Arts and Science, Iwate University, Iwate, 020-8551, Japan; Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, Iwate, 020-8551, Japan.
| | - Shungo Tanaka
- Division of Science and Engineering, Graduate School of Arts and Science, Iwate University, Iwate, 020-8551, Japan
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Yao XF, Wu S, Guo L, Liu CM. Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events. PLANT SCIENCE : AN INTERNATIONAL JOURNAL OF EXPERIMENTAL PLANT BIOLOGY 2020; 296:110469. [PMID: 32539999 DOI: 10.1016/j.plantsci.2020.110469] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/24/2019] [Revised: 03/12/2020] [Accepted: 03/14/2020] [Indexed: 06/11/2023]
Abstract
Rapid and low-cost methods of detecting mutations and polymorphisms are crucial for genotyping applications including mutagenesis and gene editing. S1 family endonucleases such as T7E1, EndoV and CELI can potentially be used in enzymatic mismatch detection. Among them, CELI has been shown to be effective in detecting mutations in Targeting Induced Local Lesions IN Genomes (TILLING). However, current method of CELI purification from celery is laborious, and challenging for many non-biochemical laboratories, and the presence of post-translational modifications hinders efficient production of the enzyme in E. coli. Here, we report an efficient system for bulk production of enzymatically active CELI endonuclease through transient expression in a model plant Nicotiana benthamiana. We also optimized the reaction buffer, by additions of Mn2+ and DTT, with enhanced mismatch cleavage activity. Using the new CELI production and reaction system, we were able to routinely detect mismatches in 1/32 mixed mutant and wildtype DNA samples. We believe the newly established system has many applications in characterization of mutations occurred in natural variations, mutagenized populations and gene editing.
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Affiliation(s)
- Xue-Feng Yao
- Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shengyang Wu
- Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China
| | - Lei Guo
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chun-Ming Liu
- Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
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The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing. Sci Rep 2020; 10:4173. [PMID: 32144373 PMCID: PMC7060192 DOI: 10.1038/s41598-020-61154-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2019] [Accepted: 02/18/2020] [Indexed: 11/08/2022] Open
Abstract
Engineered nucleases are widely used for creating frameshift or nonsense mutations in the target genes to eliminate gene functions. The resulting mRNAs carrying premature termination codons can be eliminated by nonsense-mediated mRNA decay. However, it is unclear how effective this process would be in vivo. Here, we found that the nonsense-mediated decay was unable to remove the mutant mRNAs in twelve out of sixteen homozygous mutant mice with frameshift mutations generated using engineered nucleases, which is far beyond what we expected. The frameshift mutant proteins translated by a single nucleotide deletion within the coding region were also detected in the p53 mutant mice. Furthermore, we showed that targeting the exons present downstream of the exons with a start codon or distant from ATG is relatively effective for eliminating mutant mRNAs in vivo, whereas the exons with a start codon are targeted to express the mutant mRNAs. Of the sixteen mutant mice generated, only four mutant mice targeting the downstream exons exhibited over 80% clearance of mutant mRNAs. Since the abnormal products, either mutant RNAs or mutant proteins, expressed by the target alleles might obscure the outcome of genome editing, these findings will provide insights in the improved performance of engineered nucleases when they are applied in vivo.
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Kim GD, Lee JH, Song S, Kim SW, Han JS, Shin SP, Park BC, Park TS. Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase. FASEB J 2020; 34:5688-5696. [PMID: 32100378 DOI: 10.1096/fj.201903035r] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2019] [Revised: 01/27/2020] [Accepted: 02/16/2020] [Indexed: 12/20/2022]
Abstract
Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off-target effects, including the cleavage of unintended targets, limits the practical applications of Cas9-mediated genome editing. In this study, to minimize the off-target effects of this technology, we utilized D10A-Cas9 nickase to generate myostatin-knockout (MSTN KO) chickens via primordial germ cells. D10A-Cas9 nickase (Cas9n)-mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild-type chickens. Our results demonstrate that the D10A-Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome-edited chickens in practical industrialization and as animal models of human diseases.
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Affiliation(s)
- Gap-Don Kim
- Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, South Korea.,Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang-gun, South Korea
| | - Jeong Hyo Lee
- Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang-gun, South Korea
| | - Sumin Song
- Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, South Korea
| | - Seo Woo Kim
- Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, South Korea
| | - Ji Seon Han
- Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, South Korea
| | - Seung Pyo Shin
- Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang-gun, South Korea
| | - Byung-Chul Park
- Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, South Korea.,Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang-gun, South Korea.,Research Institute of Agriculture and Life Science, Seoul National University, Seoul, South Korea
| | - Tae Sub Park
- Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, South Korea.,Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang-gun, South Korea
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Hirotsune S, Kiyonari H, Jin M, Kumamoto K, Yoshida K, Shinohara M, Watanabe H, Wynshaw-Boris A, Matsuzaki F. Enhanced homologous recombination by the modulation of targeting vector ends. Sci Rep 2020; 10:2518. [PMID: 32054870 PMCID: PMC7018964 DOI: 10.1038/s41598-020-58893-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Accepted: 12/11/2019] [Indexed: 11/17/2022] Open
Abstract
The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.
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Affiliation(s)
- Shinji Hirotsune
- Department of Genetic Disease Research, Osaka City University, Graduate School of Medicine, Asahi-machi 1-4-3, Abeno, Osaka, 545-8585, Japan.
| | - Hiroshi Kiyonari
- Animal Resource Development Unit, Genetic Engineering Team, Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-Minamimachi, Chuou-ku, Kobe, 650-0047, Japan
| | - Mingyue Jin
- Department of Genetic Disease Research, Osaka City University, Graduate School of Medicine, Asahi-machi 1-4-3, Abeno, Osaka, 545-8585, Japan
| | - Kanako Kumamoto
- Department of Genetic Disease Research, Osaka City University, Graduate School of Medicine, Asahi-machi 1-4-3, Abeno, Osaka, 545-8585, Japan
| | - Kayo Yoshida
- Laboratory Animal Science, Osaka City University, Graduate School of Medicine, Asahi-machi 1-4-3, Abeno, Osaka, 545-8585, Japan
| | - Miki Shinohara
- Institute for Protein Research, Osaka University 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
- Faculty of Agriculture, Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara-city, Nara, 631-8505, Japan
| | - Hitomi Watanabe
- Laboratory of Integrative Biological Science, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan
| | - Anthony Wynshaw-Boris
- Department of Genetics and Genome Sciences, Case Western Reserve University, School of Medicine, University Hospitals Case Medical Center 10900 Euclid Avenue, BRB731, Cleveland, Ohio, 44106-4955, USA
| | - Fumio Matsuzaki
- RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuou-ku, Kobe, 650-0047, Japan
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Fan C, Gao Y, Liang G, Huang L, Wang J, Yang X, Shi Y, Dräger UC, Zhong M, Gao TM, Yang X. Transcriptomics of Gabra4 knockout mice reveals common NMDAR pathways underlying autism, memory, and epilepsy. Mol Autism 2020; 11:13. [PMID: 32033586 PMCID: PMC7007694 DOI: 10.1186/s13229-020-0318-9] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2019] [Accepted: 01/26/2020] [Indexed: 12/16/2022] Open
Abstract
Autism spectrum disorder (ASD) is a neuronal developmental disorder with impaired social interaction and communication, often with abnormal intelligence and comorbidity with epilepsy. Disturbances in synaptic transmission, including the GABAergic, glutamatergic, and serotonergic systems, are known to be involved in the pathogenesis of this disorder, yet we do not know if there is a common molecular mechanism. As mutations in the GABAergic receptor subunit gene GABRA4 are reported in patients with ASD, we eliminated the Gabra4 gene in mice and found that the Gabra4 knockout mice showed autistic-like behavior, enhanced spatial memory, and attenuated susceptibility to pentylenetetrazol-induced seizures, a constellation of symptoms resembling human high-functioning autism. To search for potential molecular pathways involved in these phenotypes, we performed a hippocampal transcriptome profiling, constructed a hippocampal interactome network, and revealed an upregulation of the NMDAR system at the center of the converged pathways underlying high-functioning autism-like and anti-epilepsy phenotypes.
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Affiliation(s)
- Cuixia Fan
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Institute of Neuroscience and Department of Neurology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, China
| | - Yue Gao
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, 510515, China.,Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, 1838 N. Guangzhou Ave, Guangzhou, 510515, China
| | - Guanmei Liang
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, 510515, China.,Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, 1838 N. Guangzhou Ave, Guangzhou, 510515, China
| | - Lang Huang
- Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, 510515, China.,State Key Laboratory of Organ Failure Research, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Jing Wang
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Xiaoxue Yang
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Yiwu Shi
- Institute of Neuroscience and Department of Neurology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, China
| | - Ursula C Dräger
- Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA, 01655, USA
| | - Mei Zhong
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Tian-Ming Gao
- Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, 510515, China.,State Key Laboratory of Organ Failure Research, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Xinping Yang
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China. .,Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, 510515, China. .,Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, 1838 N. Guangzhou Ave, Guangzhou, 510515, China.
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Kim YS, Kim GR, Park M, Yang SC, Park SH, Won JE, Lee JH, Shin HE, Song H, Kim HR. Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor. Tissue Eng Regen Med 2020; 17:45-53. [PMID: 32002841 PMCID: PMC6992802 DOI: 10.1007/s13770-019-00225-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2019] [Revised: 10/15/2019] [Accepted: 10/16/2019] [Indexed: 01/24/2023] Open
Abstract
BACKROUND CRISPR/Cpf1 is a class II, type V RNA-guided endonuclease that is distinct from the type II CRISPR/Cas9 nuclease, widely used for genome editing. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR/Cas9 system. The applications of CRISPR to rodent embryos for the production of knock-out (KO) mice have been achieved mainly by microinjection, which requires heavily-equipped instruments with skillful hands. Here, we evaluated the genome editing efficiency between Cpf1/mRNA and Cpf1/ribonuclear protein (RNP) in mouse embryos, and established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system. METHODS The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined. RESULTS Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting. CONCLUSION Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.
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Affiliation(s)
- Yeon Sun Kim
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
| | - Gyeong Ryeong Kim
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
| | - Mira Park
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
| | - Seung Chel Yang
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
| | - So Hee Park
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
| | - Ji Eun Won
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
| | - Ju Hee Lee
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
| | - Ha Eun Shin
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
| | - Haengseok Song
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea.
| | - Hye-Ryun Kim
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea.
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Bacman SR, Gammage PA, Minczuk M, Moraes CT. Manipulation of mitochondrial genes and mtDNA heteroplasmy. Methods Cell Biol 2020; 155:441-487. [PMID: 32183972 DOI: 10.1016/bs.mcb.2019.12.004] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences. We have taken advantage of these enzymes to develop reagents to selectively eliminate mutant mtDNA. The replication of intact mitochondrial genomes normalizes mtDNA levels and consequently mitochondrial function. In this chapter, we describe the methodology used to design and express these nucleases in mammalian cells in culture and in vivo.
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Affiliation(s)
- Sandra R Bacman
- Department of Neurology, University of Miami School of Medicine, Miami, FL, United States
| | - P A Gammage
- CRUK Beatson Institute for Cancer Research, Glasgow, United Kingdom
| | - M Minczuk
- MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom.
| | - Carlos T Moraes
- Department of Neurology, University of Miami School of Medicine, Miami, FL, United States.
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Abstract
A transgenic mouse carries within its genome an artificial DNA construct (transgene) that is deliberately introduced by an experimentalist. These animals are widely used to understand gene function and protein function. When addressing the history of transgenic mouse technology, it is apparent that a number of basic science research areas laid the groundwork for success. These include reproductive science, genetics and molecular biology, and micromanipulation and microscopy equipment. From reproductive physiology came applications on how to optimize mouse breeding, how to superovulate mice to produce zygotes for DNA microinjection or preimplantation embryos for combination with embryonic stem (ES) cells, and how to return zygotes and embryos to a pseudopregnant surrogate dam for gestation and birth. From developmental biology, it was learned how to micromanipulate embryos for morula aggregation and blastocyst microinjection and how to establish germline competent ES cells. From genetics came the foundational principles governing the inheritance of genes, the interactions of gene products, and an understanding of the phenotypic consequences of genetic mutations. From molecular biology came a panoply of tools and reagents that are used to clone DNA transgenes, to detect the presence of transgenes, to assess gene expression by measuring transcription, and to detect proteins in cells and tissues. Technical advances in light microscopes, micromanipulators, micropipette pullers, and ancillary equipment made it possible for experimentalists to insert thin glass needles into zygotes or embryos under controlled conditions to inject DNA solutions or ES cells. To fully discuss the breadth of contributions of these numerous scientific disciplines to a comprehensive history of transgenic science is beyond the scope of this work. Examples will be used to illustrate scientific developments central to the foundation of transgenic technology and that are in use today.
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Affiliation(s)
- Thomas L Saunders
- Transgenic Animal Model Core, University of Michigan Medical School, Ann Arbor, MI, USA.
- Division of Genetic Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
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47
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Zheng N, Li L, Wang X. Molecular mechanisms, off-target activities, and clinical potentials of genome editing systems. Clin Transl Med 2020; 10:412-426. [PMID: 32508055 PMCID: PMC7240848 DOI: 10.1002/ctm2.34] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 04/21/2020] [Accepted: 04/21/2020] [Indexed: 12/13/2022] Open
Abstract
Methodologies of genome editing are rapidly developing with the improvement of gene science and technology, mechanism-based understanding, and urgent needs. In addition to the specificity and efficiency of on-target sites, one of the most important issues is to find and avoid off-targets before clinical application of gene editing as a therapy. Various algorithms, modified nucleases, and delivery vectors are developed to localize and minimize off-target sites. The present review aimed to clarify off-targets of various genome editing and explore potentials of clinical application by understanding structures, mechanisms, clinical applications, and off-target activities of genome editing systems, including CRISPR/Cas9, CRISPR/Cas12a, zinc finger nucleases, transcription activator-like effector nucleases, meganucleases, and recent developments. Current genome editing in cancer therapy mainly targeted immune systems in tumor microenvironment by ex vivo modification of the immune cells in phases I/II of clinical trials. We believe that genome editing will be the critical part of clinical precision medicine strategy and multidisciplinary therapy strategy by integrating gene sequencing, clinical transomics, and single cell biomedicine. There is an urgent need to develop on/off-target-specific biomarkers to monitor the efficacy and side-effects of gene therapy. Thus, the genome editing will be an alternative of clinical therapies for cancer with the rapid development of methodology and an important part of clinical precision medicine strategy.
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Affiliation(s)
- Nannan Zheng
- Zhongshan Hospital Institute for Clinical ScienceShanghai Institute of Clinical BioinformaticsShanghai Engineering Research for AI Technology for Cardiopulmonary DiseasesFudan UniversityShanghaiChina
| | - Liyang Li
- Zhongshan Hospital Institute for Clinical ScienceShanghai Institute of Clinical BioinformaticsShanghai Engineering Research for AI Technology for Cardiopulmonary DiseasesFudan UniversityShanghaiChina
| | - Xiangdong Wang
- Zhongshan Hospital Institute for Clinical ScienceShanghai Institute of Clinical BioinformaticsShanghai Engineering Research for AI Technology for Cardiopulmonary DiseasesFudan UniversityShanghaiChina
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48
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Wang A, Wang R, Cui D, Huang X, Yuan L, Liu H, Fu Y, Liang L, Wang W, He Q, Shi C, Guan X, Teng Z, Zhao G, Li Y, Gao Y, Han H. The Drainage of Interstitial Fluid in the Deep Brain is Controlled by the Integrity of Myelination. Aging Dis 2019; 10:937-948. [PMID: 31595193 PMCID: PMC6764732 DOI: 10.14336/ad.2018.1206] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2018] [Accepted: 10/26/2018] [Indexed: 12/17/2022] Open
Abstract
In searching for the drainage route of the interstitial fluid (ISF) in the deep brain, we discovered a regionalized ISF drainage system as well as a new function of myelin in regulating the drainage. The traced ISF from the caudate nucleus drained to the ipsilateral cortex along myelin fiber tracts, while in the opposite direction, its movement to the adjacent thalamus was completely impeded by a barrier structure, which was identified as the converged, compact myelin fascicle. The regulating and the barrier effects of myelin were unchanged in AQP4-knockout rats but were impaired as the integrity of boundary structure of drainage system was destroyed in a demyelinated rat model. We thus proposed that the brain homeostasis was maintained within each ISF drainage division locally, rather than across the brain as a whole. A new brain division system and a new pathogenic mechanism of demyelination are therefore proposed.
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Affiliation(s)
- Aibo Wang
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Rui Wang
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Dehua Cui
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Xinrui Huang
- Department of Biophysics, School of Basic Medical Sciences, Peking University, Beijing, China.
| | - Lan Yuan
- Peking University Medical and Health Analysis Center, Peking University Health Science Center, Beijing, China.
| | - Huipo Liu
- Institute of Applied Physics and Computational Mathematics, Beijing, China.
| | - Yu Fu
- Department of Neurology, Peking University Third Hospital, Beijing, China.
| | - Lei Liang
- Department of Medical Chemistry, School of Pharmaceutical Sciences, Peking University, Beijing, China.
| | - Wei Wang
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Qingyuan He
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Chunyan Shi
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Xiangping Guan
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Ze Teng
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Guomei Zhao
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Yuanyuan Li
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Yajuan Gao
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
| | - Hongbin Han
- Department of Radiology, Peking University Third Hospital, Beijing, China.
- Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China.
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Yeo JH, Jung BK, Lee H, Baek IJ, Sung YH, Shin HS, Kim HK, Seo KY, Lee JY. Development of a Pde6b Gene Knockout Rat Model for Studies of Degenerative Retinal Diseases. Invest Ophthalmol Vis Sci 2019; 60:1519-1526. [PMID: 31009522 DOI: 10.1167/iovs.18-25556] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Purpose To describe the phenotypes of a newly developed Pde6b-deficient rat model of retinal degeneration. Methods Pde6b knockout rats were produced by CRISPR-Cpf1 technology. Pde6b knockout rats were evaluated for ocular abnormalities by comparison with wild-type eyes. Eyes were imaged using fundus photography and optical coherence tomography (OCT), stained by hematoxylin and eosin (H&E), and examined by TUNEL assay. Finally, eyes were functionally assessed by electroretinograms (ERGs). Results Pde6b knockout rats exhibited visible photoreceptor degeneration at 3 weeks of postnatal age. The fundus appearance of mutants was notable for pigmentary changes, vascular attenuation with an irregular vascular pattern, and outer retinal thinning, which resembled retinitis pigmentosa (RP) in humans. OCT showed profound retinal thinning in Pde6b knockout rats; the outer nuclear layer (ONL) was significantly thinner in Pde6b knockout rats, with relative preservation of the inner retina at 3 weeks of postnatal age. H&E staining confirmed extensive degeneration of the ONL, beginning at 3 weeks of postnatal age; no ONL remained in the retina by 16 weeks of postnatal age. Retinal sections of Pde6b knockout rats were highly positive for TUNEL, specifically in the ONL. In ERGs, Pde6b knockout rats showed no detectable a- or b-waves at 8 weeks of postnatal age. Conclusions The Pde6b knockout rat exhibits photoreceptor degeneration. It may provide a better model for experimental therapy for RP because of its slower progression and larger anatomic architecture than the corresponding mouse model. Further studies in this rat model may yield insights into effective therapies for human RP.
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Affiliation(s)
- Joon Hyung Yeo
- Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Bok Kyoung Jung
- Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.,Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea
| | - Heuiran Lee
- Department of Microbiology, University of Ulsan College of Medicine, Seoul, Korea.,Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea
| | - In-Jeoung Baek
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea.,Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul, Korea
| | - Young Hoon Sung
- Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea.,Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul, Korea
| | - Hae-Sol Shin
- Korea Mouse Sensory Phenotyping Center (KMSPC), Yonsei University College of Medicine, Seoul, Korea.,Institute for Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea
| | - Hong Kyung Kim
- Korea Mouse Sensory Phenotyping Center (KMSPC), Yonsei University College of Medicine, Seoul, Korea.,Institute for Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea
| | - Kyoung Yul Seo
- Korea Mouse Sensory Phenotyping Center (KMSPC), Yonsei University College of Medicine, Seoul, Korea.,Institute for Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea
| | - Joo Yong Lee
- Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.,Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea
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50
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Shin MG, Cha HN, Park S, Kim YW, Kim JY, Park SY. Selenoprotein W deficiency does not affect oxidative stress and insulin sensitivity in the skeletal muscle of high-fat diet-fed obese mice. Am J Physiol Cell Physiol 2019; 317:C1172-C1182. [PMID: 31509445 DOI: 10.1152/ajpcell.00064.2019] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Selenoprotein W (SelW) is a selenium-containing protein with a redox motif found abundantly in the skeletal muscle of rodents. Previous in vitro studies suggest that SelW plays an antioxidant role; however, relatively few in vivo studies have addressed the antioxidant role of SelW. Since oxidative stress is a causative factor for the development of insulin resistance in obese subjects, we hypothesized that if SelW plays a role as an antioxidant, SelW deficiency could aggravate the oxidative stress and insulin resistance caused by a high-fat diet. SelW deficiency did not affect insulin sensitivity and H2O2 levels in the skeletal muscle of control diet-fed mice. SelW levels in the skeletal muscle were decreased by high-fat diet feeding for 12 wk. High-fat diet induced obesity and insulin resistance and increased the levels of H2O2 and oxidative stress makers, which were not affected by SelW deficiency. High-fat diet feeding increased the expression of antioxidant enzymes; however, SelW deficiency did not affect the expression levels of antioxidants. These results suggest that SelW does not play a protective role against oxidative stress and insulin resistance in the skeletal muscle of high-fat diet-fed obese mice.
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Affiliation(s)
- Min-Gyeong Shin
- Department of Physiology, College of Medicine, Yeungnam University, Daegu, Korea.,Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, Daegu, Korea
| | - Hye-Na Cha
- Department of Physiology, College of Medicine, Yeungnam University, Daegu, Korea.,Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, Daegu, Korea
| | - Soyoung Park
- Department of Physiology, College of Medicine, Yeungnam University, Daegu, Korea.,Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, Daegu, Korea
| | - Yong-Woon Kim
- Department of Physiology, College of Medicine, Yeungnam University, Daegu, Korea
| | - Jong-Yeon Kim
- Department of Physiology, College of Medicine, Yeungnam University, Daegu, Korea
| | - So-Young Park
- Department of Physiology, College of Medicine, Yeungnam University, Daegu, Korea.,Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, Daegu, Korea
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