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1
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In vivo enrichment of busulfan-resistant germ cells for efficient production of transgenic avian models. Sci Rep 2021; 11:9127. [PMID: 33911174 PMCID: PMC8080772 DOI: 10.1038/s41598-021-88706-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Accepted: 04/16/2021] [Indexed: 01/01/2023] Open
Abstract
Most transgenic animals are generated using a genome-modified stem cell system and genome modification directly in embryos. Although this system is well-established in the development of transgenic animals, donor cell-derived transgenic animal production is inefficient in some cases. Especially in avian models such as chickens, the efficiency of transgenic animal production through primordial germ cells (PGCs) is highly variable compared with embryonic manipulation of mammalian species. Because germ cell and germline-competent stem cell-mediated systems that contain the transgene are enriched only at the upstream level during cell cultivation, the efficiency of transgenic animal production is unreliable. Therefore, we developed an in vivo selection model to enhance the efficiency of transgenic chicken production using microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to the alkylating agent busulfan, which induces germ cell-specific cytotoxicity. Under in vitro conditions, MGSTII-tg PGCs were resistant to 1 μM busulfan, which was highly toxic to wild-type PGCs. In germline chimeric roosters, transgene-expressing germ cells were dominantly colonized in the recipient testes after busulfan exposure compared with non-treated germline chimera. In validation of germline transmission, donor PGC-derived progeny production efficiency was 94.68%, and the transgene production rate of heterozygous transgenic chickens was significantly increased in chickens that received 40 mg/kg busulfan (80.33–95.23%) compared with that of non-treated germline chimeras (51.18%). This system is expected to significantly improve the efficiency of generating transgenic chickens and other animal species by increasing the distribution of donor cells in adult testes.
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2
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Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response. Nat Commun 2020; 11:2396. [PMID: 32409666 PMCID: PMC7224190 DOI: 10.1038/s41467-020-16271-z] [Citation(s) in RCA: 69] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Accepted: 04/20/2020] [Indexed: 02/07/2023] Open
Abstract
Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response. Protein arginine methyltransferases (PRMTs) are increasingly recognized as potential therapeutic targets but PRMT7 remains an understudied member of this enzyme family. Here, the authors develop a chemical probe for PRMT7 and apply it to elucidate the role of PRMT7 in the cellular stress response.
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3
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Abstract
Transgenic technology in rats is increasingly important for the design and implementation of biological and physiological studies in the fields of neuroscience, pharmacology, and toxicology. Pluripotent embryonic stem cells (ESCs) are a useful tool for generation of gene-modified rats. During the last decade, not only foreign DNA introduction but also endogenous DNA modification has been successfully achieved with rat ESCs. Detailed protocols for establishment of bona fide rat ESCs and their use for production of gene-modified rats are described in this chapter.
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4
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Hirabayashi M, Hochi S. Organ Generation from Knockedout Rat Blastocysts Complemented with Pluripotent Stem Cells. Methods Mol Biol 2019; 1874:313-326. [PMID: 30353522 DOI: 10.1007/978-1-4939-8831-0_18] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Regeneration of human organs in domestic animal model would provide enough number of functional donor organs in transplantation therapy. Recent progresses in pluripotent stem cells and nuclease-based genome editing tools have set the stage for investigating the chimeric complementation approach to generate functional organs from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. In this chapter, protocol for allogeneic or xenogeneic organ generation using knocked-out (KO) rat blastocysts and the rat or mouse ES/iPS cells is described. The protocol includes (1) the preparation of KO rat colony, (2) the preparation of rat or mouse ES/iPS cells, (3) the recovery of rat blastocysts, (4) the stem cell injection into blastocysts, (5) the embryo transfer into pseudopregnant recipient uteri, and (6) the genotyping and organogenetic analysis of chimeric offspring. The accumulation of basic and practical knowledge in the rodent model would be useful in improving therapeutic performance to regenerate 3D organs available for transplantation.
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Affiliation(s)
- Masumi Hirabayashi
- The Graduate University for Advanced Studies, Okazaki, Aichi, Japan. .,Section of Mammalian Transgenesis, Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.
| | - Shinichi Hochi
- Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan
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5
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Abstract
The laboratory rat, Rattus norvegicus, has been used in biomedical research for more than 150 years, and in many cases remains the model of choice for studies of physiology, behavior, and complex human disease. This book provides detailed information on a number of methodologies that can be used in rat. This chapter gives an introduction to rat as a species and as a biomedical model, providing historical information, a brief introduction to the current state of rat research, and a perspective on the future of rat as a model for human disease.
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Affiliation(s)
- Jennifer R Smith
- Department of Biomedical Engineering, Rat Genome Database, Medical College of Wisconsin, Milwaukee, WI, USA.
| | - Elizabeth R Bolton
- Department of Biomedical Engineering, Rat Genome Database, Medical College of Wisconsin, Milwaukee, WI, USA
| | - Melinda R Dwinell
- Genomic Sciences and Precision Medicine Center, Medical College of Wisconsin, Milwaukee, WI, USA
- Department of Physiology, Rat Genome Database, Medical College of Wisconsin, Milwaukee, WI, USA
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6
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Abstract
Since its domestication over 100 years ago, the laboratory rat has been the preferred experimental animal in many areas of biomedical research (Lindsey and Baker The laboratory rat. Academic, New York, pp 1-52, 2006). Its physiology, size, genetics, reproductive cycle, cognitive and behavioural characteristics have made it a particularly useful animal model for studying many human disorders and diseases. Indeed, through selective breeding programmes numerous strains have been derived that are now the mainstay of research on hypertension, obesity and neurobiology (Okamoto and Aoki Jpn Circ J 27:282-293, 1963; Zucker and Zucker J Hered 52(6):275-278, 1961). Despite this wealth of genetic and phenotypic diversity, the ability to manipulate and interrogate the genetic basis of existing phenotypes in rat strains and the methodology to generate new rat models has lagged significantly behind the advances made with its close cousin, the laboratory mouse. However, recent technical developments in stem cell biology and genetic engineering have again brought the rat to the forefront of biomedical studies and enabled researchers to exploit the increasingly accessible wealth of genome sequence information. In this review, we will describe how a breakthrough in understanding the molecular basis of self-renewal of the pluripotent founder cells of the mammalian embryo, embryonic stem (ES) cells, enabled the derivation of rat ES cells and their application in transgenesis. We will also describe the remarkable progress that has been made in the development of gene editing enzymes that enable the generation of transgenic rats directly through targeted genetic modifications in the genomes of zygotes. The simplicity, efficiency and cost-effectiveness of the CRISPR/Cas gene editing system, in particular, mean that the ability to engineer the rat genome is no longer a limiting factor. The selection of suitable targets and gene modifications will now become a priority: a challenge where ES culture and gene editing technologies can play complementary roles in generating accurate bespoke rat models for studying biological processes and modelling human disease.
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7
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Lan H, Li S, Guo Z, Men H, Wu Y, Li N, Bryda EC, Capecchi MR, Wu S. Efficient generation of selection-gene-free rat knockout models by homologous recombination in ES cells. FEBS Lett 2016; 590:3416-3424. [PMID: 27597178 PMCID: PMC5129459 DOI: 10.1002/1873-3468.12388] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2016] [Revised: 08/24/2016] [Accepted: 08/25/2016] [Indexed: 11/25/2022]
Abstract
Embryonic stem cell (ES cell)‐based rat knockout technology, although successfully developed in 2010, has seen very limited usage to date due to low targeting efficiency and a lack of optimized procedures. In this study, we performed gene targeting in ES cells from the Sprague–Dawley (SD) and the Fischer 344 (F344) rat strains using an optimized procedure and the self‐excising neomycin (neo)‐positive selection cassette ACN to successfully generate Leptin and Trp53 knockout rats that did not carry the selection gene. These results demonstrate that our simplified targeting strategy using ACN provides an efficient approach to knock out many other rat genes.
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Affiliation(s)
- He Lan
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Shuping Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Zihang Guo
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Hongsheng Men
- Veterinary Pathobiology, Rat Resource and Research Center, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA
| | - Yuanyuan Wu
- Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA
| | - Ning Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Elizabeth C Bryda
- Veterinary Pathobiology, Rat Resource and Research Center, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA
| | - Mario R Capecchi
- Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA
| | - Sen Wu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
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8
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Tesson L, Remy S, Ménoret S, Usal C, Thinard R, Savignard C, De Cian A, Giovannangeli C, Concordet JP, Anegon I. Genome Editing in Rats Using TALE Nucleases. Methods Mol Biol 2016; 1338:245-59. [PMID: 26443226 DOI: 10.1007/978-1-4939-2932-0_18] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.
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Affiliation(s)
- Laurent Tesson
- Transgenic Rats Nantes IBiSA - Centre National de Recherche Scientifique, 44093, Nantes, France. .,ITUN, CHU Nantes, 30 Bvd J. Monnet, 44093, Nantes, France. .,INSERM UMR 1064, Center for Research in Transplantation and Immunology, Nantes, France.
| | - Séverine Remy
- Transgenic Rats Nantes IBiSA - Centre National de Recherche Scientifique, 44093, Nantes, France.,ITUN, CHU Nantes, 30 Bvd J. Monnet, 44093, Nantes, France.,INSERM UMR 1064, Center for Research in Transplantation and Immunology, Nantes, France
| | - Séverine Ménoret
- Transgenic Rats Nantes IBiSA - Centre National de Recherche Scientifique, 44093, Nantes, France.,ITUN, CHU Nantes, 30 Bvd J. Monnet, 44093, Nantes, France.,INSERM UMR 1064, Center for Research in Transplantation and Immunology, Nantes, France
| | - Claire Usal
- Transgenic Rats Nantes IBiSA - Centre National de Recherche Scientifique, 44093, Nantes, France.,ITUN, CHU Nantes, 30 Bvd J. Monnet, 44093, Nantes, France.,INSERM UMR 1064, Center for Research in Transplantation and Immunology, Nantes, France
| | - Reynald Thinard
- Transgenic Rats Nantes IBiSA - Centre National de Recherche Scientifique, 44093, Nantes, France.,ITUN, CHU Nantes, 30 Bvd J. Monnet, 44093, Nantes, France.,INSERM UMR 1064, Center for Research in Transplantation and Immunology, Nantes, France
| | - Chloé Savignard
- Transgenic Rats Nantes IBiSA - Centre National de Recherche Scientifique, 44093, Nantes, France.,ITUN, CHU Nantes, 30 Bvd J. Monnet, 44093, Nantes, France.,INSERM UMR 1064, Center for Research in Transplantation and Immunology, Nantes, France
| | - Anne De Cian
- INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, 43 rue Cuvier, 75005, Paris, France
| | - Carine Giovannangeli
- INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, 43 rue Cuvier, 75005, Paris, France
| | - Jean-Paul Concordet
- INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, 43 rue Cuvier, 75005, Paris, France
| | - Ignacio Anegon
- Transgenic Rats Nantes IBiSA - Centre National de Recherche Scientifique, 44093, Nantes, France.,ITUN, CHU Nantes, 30 Bvd J. Monnet, 44093, Nantes, France.,INSERM UMR 1064, Center for Research in Transplantation and Immunology, Nantes, France
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9
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Meek S, Thomson AJ, Sutherland L, Sharp MGF, Thomson J, Bishop V, Meddle SL, Gloaguen Y, Weidt S, Singh-Dolt K, Buehr M, Brown HK, Gill AC, Burdon T. Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease. Sci Rep 2016; 6:25592. [PMID: 27185277 PMCID: PMC4869022 DOI: 10.1038/srep25592] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2016] [Accepted: 04/19/2016] [Indexed: 02/07/2023] Open
Abstract
Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour.
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Affiliation(s)
- Stephen Meek
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland
| | - Alison J. Thomson
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland
| | - Linda Sutherland
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland
| | - Matthew G. F. Sharp
- Central Bioresearch Services, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland
| | - Julie Thomson
- Central Bioresearch Services, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland
| | - Valerie Bishop
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland
| | - Simone L. Meddle
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland
| | - Yoann Gloaguen
- Glasgow Polyomics, College of Medical, Veterinary and Life Sciences, University of Glasgow, Wolfson Wohl Cancer Research Centre, Garscube Campus, Bearsden, G61 1QH, Scotland
| | - Stefan Weidt
- Glasgow Polyomics, College of Medical, Veterinary and Life Sciences, University of Glasgow, Wolfson Wohl Cancer Research Centre, Garscube Campus, Bearsden, G61 1QH, Scotland
| | - Karamjit Singh-Dolt
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland
| | - Mia Buehr
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland
| | - Helen K. Brown
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland
| | - Andrew C. Gill
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland,
| | - Tom Burdon
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland,
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10
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Generation of Hprt-disrupted rat through mouse←rat ES chimeras. Sci Rep 2016; 6:24215. [PMID: 27062982 PMCID: PMC4827079 DOI: 10.1038/srep24215] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2016] [Accepted: 03/22/2016] [Indexed: 11/08/2022] Open
Abstract
We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse←rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse←rat ES chimera system. The mouse←rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production.
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11
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Yamamoto S, Ooshima Y, Nakata M, Yano T, Nishimura N, Nishigaki R, Satomi Y, Matsumoto H, Matsumoto Y, Takeyama M. Efficient gene-targeting in rat embryonic stem cells by CRISPR/Cas and generation of human kynurenine aminotransferase II (KAT II) knock-in rat. Transgenic Res 2015; 24:991-1001. [PMID: 26454650 DOI: 10.1007/s11248-015-9909-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2015] [Accepted: 10/06/2015] [Indexed: 12/26/2022]
Abstract
The relative proportion of kynurenine aminotransferase (KAT) I-IV activities in the brain is similar between humans and rats. Moreover, KAT II is considered to be the main enzyme for kynurenic acid production in the brain. Taken together, human KAT II knock-in (hKAT II KI) rats will become a valuable tool for the evaluation of KAT II targeted drugs as a human mimetic model. Although we initially tried the approach by conventional gene-targeting via embryonic stem cells (ESCs) to generate them, we had to give up the production because of no recombinant ESCs. Accordingly, we developed a method to improve the efficiency of homologous recombination (HR) in ESCs by the combination with the CRISPR/Cas system. Co-electroporation of Cas9 plasmid, single guide RNA plasmid and hKAT II KI vector increased the number of drug-resistant colonies and greatly enhanced the HR efficiency from 0 to 36 %. All the clones which we obtained showed the same sequence as designed. These recombinant clones resulted in chimeras that transmitted the hKAT II KI allele to their offspring. hKAT II KI rats showed no reduction of KATs mRNA expression and the amount of kynurenic acid was similar between the hKAT II KI rats and the wild type in their brains. These results indicate that the methodology presented in this report can overcome the problem encountered in conventional gene-targeting that prevented production of humanized rats.
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Affiliation(s)
- Satoshi Yamamoto
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
| | - Yuki Ooshima
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan
| | - Mitsugu Nakata
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan
| | - Takashi Yano
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan
| | - Naoya Nishimura
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan
| | - Ryuuichi Nishigaki
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan
| | - Yoshinori Satomi
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan
| | - Hirokazu Matsumoto
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan
| | - Yoshio Matsumoto
- Takeda Rabics Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan
| | - Michiyasu Takeyama
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
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12
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Kawaharada K, Kawamata M, Ochiya T. Rat embryonic stem cells create new era in development of genetically manipulated rat models. World J Stem Cells 2015; 7:1054-1063. [PMID: 26328021 PMCID: PMC4550629 DOI: 10.4252/wjsc.v7.i7.1054] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/22/2014] [Revised: 01/15/2015] [Accepted: 07/17/2015] [Indexed: 02/07/2023] Open
Abstract
Embryonic stem (ES) cells are isolated from the inner cell mass of a blastocyst, and are used for the generation of gene-modified animals. In mice, the transplantation of gene-modified ES cells into recipient blastocysts leads to the creation of gene-targeted mice such as knock-in and knock-out mice; these gene-targeted mice contribute greatly to scientific development. Although the rat is considered a useful laboratory animal alongside the mouse, fewer gene-modified rats have been produced due to the lack of robust establishment methods for rat ES cells. A new method for establishing rat ES cells using signaling inhibitors was reported in 2008. By considering the characteristics of rat ES cells, recent research has made progress in improving conditions for the stable culture of rat ES cells in order to generate gene-modified rats efficiently. In this review, we summarize several advanced methods to maintain rat ES cells and generate gene-targeted rats.
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13
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Flister MJ, Prokop JW, Lazar J, Shimoyama M, Dwinell M, Geurts A. 2015 Guidelines for Establishing Genetically Modified Rat Models for Cardiovascular Research. J Cardiovasc Transl Res 2015; 8:269-77. [PMID: 25920443 PMCID: PMC4475456 DOI: 10.1007/s12265-015-9626-4] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/11/2015] [Accepted: 04/15/2015] [Indexed: 12/24/2022]
Abstract
The rat has long been a key physiological model for cardiovascular research, most of the inbred strains having been previously selected for susceptibility or resistance to various cardiovascular diseases (CVD). These CVD rat models offer a physiologically relevant background on which candidates of human CVD can be tested in a more clinically translatable experimental setting. However, a diverse toolbox for genetically modifying the rat genome to test molecular mechanisms has only recently become available. Here, we provide a high-level description of several strategies for developing genetically modified rat models of CVD.
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Affiliation(s)
- Michael J Flister
- Human and Molecular Genetics Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, 53226, WI, USA,
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14
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Abstract
The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.
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Affiliation(s)
- Wei Zhang
- Nanobioscience, State University of New York College of Nanoscale Science and Engineering (SUNY CNSE)
| | - Mohamed Trebak
- Nanobioscience, State University of New York College of Nanoscale Science and Engineering (SUNY CNSE);
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15
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Zschemisch NH, Eisenblätter R, Rudolph C, Glage S, Dorsch M. Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm. SPRINGERPLUS 2014; 3:588. [PMID: 25332888 PMCID: PMC4197200 DOI: 10.1186/2193-1801-3-588] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/06/2014] [Accepted: 09/22/2014] [Indexed: 01/02/2023]
Abstract
Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-β and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture.
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Affiliation(s)
- Nils-Holger Zschemisch
- Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
| | - Regina Eisenblätter
- Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
| | - Cornelia Rudolph
- Institute for Molecular and Cellular Pathology, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
| | - Silke Glage
- Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
| | - Martina Dorsch
- Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
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Doorschodt B, Teubner A, Kobayashi E, Tolba R. Promising future for the transgenic rat in transplantation research. Transplant Rev (Orlando) 2014; 28:155-62. [DOI: 10.1016/j.trre.2014.05.002] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2014] [Revised: 04/02/2014] [Accepted: 05/20/2014] [Indexed: 01/17/2023]
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Remy S, Tesson L, Menoret S, Usal C, De Cian A, Thepenier V, Thinard R, Baron D, Charpentier M, Renaud JB, Buelow R, Cost GJ, Giovannangeli C, Fraichard A, Concordet JP, Anegon I. Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases. Genome Res 2014; 24:1371-83. [PMID: 24989021 PMCID: PMC4120090 DOI: 10.1101/gr.171538.113] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%–5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.
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Affiliation(s)
- Séverine Remy
- INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France
| | - Laurent Tesson
- INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France
| | - Séverine Menoret
- INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France
| | - Claire Usal
- INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France
| | - Anne De Cian
- INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France
| | - Virginie Thepenier
- INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France
| | - Reynald Thinard
- INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France
| | - Daniel Baron
- INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France
| | - Marine Charpentier
- INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France
| | - Jean-Baptiste Renaud
- INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France
| | - Roland Buelow
- Open Monoclonal Technologies, Palo Alto, California 94303, USA
| | | | - Carine Giovannangeli
- INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France
| | | | - Jean-Paul Concordet
- INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France
| | - Ignacio Anegon
- INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France
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18
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Meek S, Wei J, Sutherland L, Nilges B, Buehr M, Tomlinson SR, Thomson AJ, Burdon T. Tuning of β-catenin activity is required to stabilize self-renewal of rat embryonic stem cells. Stem Cells 2014; 31:2104-15. [PMID: 23843312 DOI: 10.1002/stem.1466] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2012] [Accepted: 05/30/2013] [Indexed: 12/20/2022]
Abstract
Stabilization of β-catenin, through inhibition of glycogen synthase kinase 3 (GSK3) activity, in conjunction with inhibition of mitogen-activated protein kinase kinase 1/2 (MEK) promotes self-renewal of naïve-type mouse embryonic stem cells (ESC). In developmentally more advanced, primed-type, epiblast stem cells, however, β-catenin activity induces differentiation. We investigated the response of rat ESCs to β-catenin signaling and found that when maintained on feeder-support cells in the presence of a MEK inhibitor alone (1i culture), the derivation efficiency, growth, karyotypic stability, transcriptional profile, and differentiation potential of rat ESC cultures was similar to that of cell lines established using both MEK and GSK3 inhibitors (2i culture). Equivalent mouse ESCs, by comparison, differentiated in identical 1i conditions, consistent with insufficient β-catenin activity. This interspecies difference in reliance on GSK3 inhibition corresponded with higher overall levels of β-catenin activity in rat ESCs. Indeed, rat ESCs displayed widespread expression of the mesendoderm-associated β-catenin targets, Brachyury and Cdx2 in 2i medium, and overt differentiation upon further increases in β-catenin activity. In contrast, mouse ESCs were resistant to differentiation at similarly elevated doses of GSK3 inhibitor. Interestingly, without feeder support, moderate levels of GSK3 inhibition were necessary to support effective growth of rat ESC, confirming the conserved role for β-catenin in ESC self-renewal. This work identifies β-catenin signaling as a molecular rheostat in rat ESC, regulating self-renewal in a dose-dependent manner, and highlights the potential importance of controlling flux in this signaling pathway to achieve effective stabilization of naïve pluripotency.
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Affiliation(s)
- Stephen Meek
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, United Kingdom
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Hirabayashi M, Goto T, Tamura C, Sanbo M, Hara H, Hochi S. Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines. J Reprod Dev 2014; 60:78-82. [PMID: 24317016 PMCID: PMC3958585 DOI: 10.1262/jrd.2013-109] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2013] [Accepted: 11/11/2013] [Indexed: 11/20/2022] Open
Abstract
This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines.
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Affiliation(s)
- Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Aichi 444-8787, Japan
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20
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Meek S, Sutherland L, Burdon T. Tuning differentiation signals for efficient propagation and in vitro validation of rat embryonic stem cell cultures. Methods Mol Biol 2014; 1212:73-85. [PMID: 25224161 DOI: 10.1007/7651_2014_121] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
The rat is one of the most commonly used laboratory animals in biomedical research and the recent isolation of genuine pluripotent rat embryonic stem (ES) cell lines has provided new opportunities for applying contemporary genetic engineering techniques to the rat and enhancing the use of this rodent in scientific research. Technical refinements that improve the stability of the rat ES cell cultures will undoubtedly further strengthen and broaden the use of these stem cells in biomedical research. Here, we describe a relatively simple and robust protocol that supports the propagation of germ line competent rat ES cells, and outline how tuning stem cell signaling using small molecule inhibitors can be used to both stabilize self-renewal of rat ES cell cultures and aid evaluation of their differentiation potential in vitro.
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Affiliation(s)
- Stephen Meek
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK,
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21
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Burbacher TM, Grant KS, Worlein J, Ha J, Curnow E, Juul S, Sackett GP. Four decades of leading-edge research in the reproductive and developmental sciences: the Infant Primate Research Laboratory at the University of Washington National Primate Research Center. Am J Primatol 2013; 75:1063-83. [PMID: 23873400 PMCID: PMC5452618 DOI: 10.1002/ajp.22175] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2013] [Revised: 05/31/2013] [Accepted: 05/31/2013] [Indexed: 12/20/2022]
Abstract
The Infant Primate Research Laboratory (IPRL) was established in 1970 at the University of Washington as a visionary project of Dr. Gene (Jim) P. Sackett. Supported by a collaboration between the Washington National Primate Research Center and the Center on Human Development and Disability, the IPRL operates under the principle that learning more about the causes of abnormal development in macaque monkeys will provide important insights into the origins and treatment of childhood neurodevelopmental disabilities. Over the past 40 years, a broad range of research projects have been conducted at the IPRL. Some have described the expression of normative behaviors in nursery-reared macaques while others have focused on important biomedical themes in child health and development. This article details the unique scientific history of the IPRL and the contributions produced by research conducted in the laboratory. Past and present investigations have explored the topics of early rearing effects, low-birth-weight, prematurity, birth injury, epilepsy, prenatal neurotoxicant exposure, viral infection (pediatric HIV), diarrheal disease, vaccine safety, and assisted reproductive technologies. Data from these studies have helped advance our understanding of both risk and resiliency in primate development. New directions of research at the IPRL include the production of transgenic primate models using our embryonic stem cell-based technology to better understand and treat heritable forms of human intellectual disabilities such as fragile X.
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Affiliation(s)
- Thomas M. Burbacher
- Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, WA, 98195 USA
- Center on Human Development and Disability, University of Washington, Seattle, WA, 98195 USA
- Washington National Primate Research Center, University of Washington, Seattle, WA 98195 USA
| | - Kimberly S. Grant
- Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, WA, 98195 USA
- Center on Human Development and Disability, University of Washington, Seattle, WA, 98195 USA
- Washington National Primate Research Center, University of Washington, Seattle, WA 98195 USA
| | - Julie Worlein
- Washington National Primate Research Center, University of Washington, Seattle, WA 98195 USA
| | - James Ha
- Washington National Primate Research Center, University of Washington, Seattle, WA 98195 USA
- Department of Psychology, School of Arts and Sciences, University of Washington, Seattle, WA, 98195 USA
| | - Eliza Curnow
- Washington National Primate Research Center, University of Washington, Seattle, WA 98195 USA
| | - Sandra Juul
- Center on Human Development and Disability, University of Washington, Seattle, WA, 98195 USA
- Department of Pediatrics, School of Medicine, University of Washington, Seattle, WA, 98195 USA
| | - Gene P. Sackett
- Center on Human Development and Disability, University of Washington, Seattle, WA, 98195 USA
- Washington National Primate Research Center, University of Washington, Seattle, WA 98195 USA
- Department of Psychology, School of Arts and Sciences, University of Washington, Seattle, WA, 98195 USA
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22
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Yang S, Takizawa A, Foeckler J, Zappa A, Gjoka M, Schilling R, Hansen C, Xu H, Kalloway S, Grzybowski M, Davis GD, Jacob HJ, Geurts AM. Derivation and genetic modification of embryonic stem cells from disease-model inbred rat strains. Stem Cells Dev 2013. [PMID: 23635087 DOI: 10.1089/scd.2012.0416] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022] Open
Abstract
The lack of rat embryonic stem cells (ESCs) and approaches for manipulation of their genomes have restricted the ability to create new genetic models and to explore the function of a single gene in complex diseases in the laboratory rat. The recent breakthrough in isolating germline-competent ESCs from rat and subsequent demonstration of gene knockout has propelled the field forward, but such tools do not yet exist for many disease-model rat strains. Here we derive new ESCs from several commonly used rat models including the Dahl Salt Sensitive (SS), the sequenced Brown Norway (BN), and Fischer (F344) rat and establish the first germline-competent ESCs from a hypertension disease model strain, the Fawn Hooded Hypertensive (FHH) rat. Genetic manipulations including transgenesis mediated by lentivirus, routine homologous recombination, and homologous recombination mediated by zinc-finger nucleases (ZFNs) were performed effectively in FHH rat ESCs. Our results showed these rat ESC lines, isolated from inner cell masses using mechanical splitting, had germline competency; the Pparg gene locus and homologous genomic region to the mouse Rosa26 locus can be targeted effectively in these rat ESCs. Furthermore, our results also demonstrated that ZFNs increased the efficiency of proper homologous recombination in FHH rat ESCs using targeting vectors with short homology arms. These rat ESC lines and advancements in genetic manipulation pave the way to novel genetic approaches in this valuable biomedical model species and for exploration of complex disease in these strains.
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Affiliation(s)
- Sheng Yang
- University of Michigan, Cardiac Surgery, Ann Arbor, Michigan, United States, Medical College of Wisconsin, Human and Molecular Genetics Center, Milwaukee, Wisconsin, United States ;
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23
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Miao X. Recent advances in the development of new transgenic animal technology. Cell Mol Life Sci 2013; 70:815-28. [PMID: 22833168 PMCID: PMC11113483 DOI: 10.1007/s00018-012-1081-7] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2012] [Revised: 06/30/2012] [Accepted: 07/03/2012] [Indexed: 12/14/2022]
Abstract
Transgenic animal technology is one of the fastest growing biotechnology areas. It is used to integrate exogenous genes into the animal genome by genetic engineering technology so that these genes can be inherited and expressed by offspring. The transgenic efficiency and precise control of gene expression are the key limiting factors in the production of transgenic animals. A variety of transgenic technologies are available. Each has its own advantages and disadvantages and needs further study because of unresolved technical and safety issues. Further studies will allow transgenic technology to explore gene function, animal genetic improvement, bioreactors, animal disease models, and organ transplantation. This article reviews the recently developed animal transgenic technologies, including the germ line stem cell-mediated method to improve efficiency, gene targeting to improve accuracy, RNA interference-mediated gene silencing technology, zinc-finger nuclease gene targeting technology and induced pluripotent stem cell technology. These new transgenic techniques can provide a better platform to develop transgenic animals for breeding new animal varieties and promote the development of medical sciences, livestock production, and other fields.
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Affiliation(s)
- Xiangyang Miao
- Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
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24
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Merkl C, Saalfrank A, Riesen N, Kühn R, Pertek A, Eser S, Hardt MS, Kind A, Saur D, Wurst W, Iglesias A, Schnieke A. Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector. PLoS One 2013; 8:e55170. [PMID: 23383095 PMCID: PMC3561372 DOI: 10.1371/journal.pone.0055170] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2012] [Accepted: 12/19/2012] [Indexed: 11/26/2022] Open
Abstract
Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.
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Affiliation(s)
- Claudia Merkl
- Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany
| | - Anja Saalfrank
- Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany
| | - Nathalie Riesen
- Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany
| | - Ralf Kühn
- Institute for Developmental Genetics, Helmholtz Center Munich, Munich, Germany
- Technische Universität München, Munich, Germany
| | - Anna Pertek
- Institute for Developmental Genetics, Helmholtz Center Munich, Munich, Germany
| | - Stefan Eser
- Klinikum Rechts der Isar II, Technische Universität München, Munich, Germany
| | | | - Alexander Kind
- Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany
| | - Dieter Saur
- Klinikum Rechts der Isar II, Technische Universität München, Munich, Germany
| | - Wolfgang Wurst
- Institute for Developmental Genetics, Helmholtz Center Munich, Munich, Germany
- Technische Universität München, Munich, Germany
- Deutsches Zentrum für neurodegenerative Erkrankungen e.V., Munich, Germany
| | - Antonio Iglesias
- Small Molecule Research - Discovery Technologies, Pharma Research and Early Development, F. Hoffmann-La Roche Ltd., Basle, Switzerland
| | - Angelika Schnieke
- Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany
- * E-mail:
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Ménoret S, Fontanière S, Jantz D, Tesson L, Thinard R, Rémy S, Usal C, Ouisse LH, Fraichard A, Anegon I. Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB J 2012; 27:703-11. [PMID: 23150522 DOI: 10.1096/fj.12-219907] [Citation(s) in RCA: 79] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Despite the recent availability of gene-specific nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like nucleases (TALENs), there is still a need for new tools to modify the genome of different species in an efficient, rapid, and less costly manner. One aim of this study was to apply, for the first time, engineered meganucleases to mutate an endogenous gene in animal zygotes. The second aim was to target the mouse and rat recombination activating gene 1 (Rag1) to describe, for the first time, Rag1 knockout immunodeficient rats. We microinjected a plasmid encoding a meganuclease for Rag1 into the pronucleus of mouse and rat zygotes. Mutant animals were detected by PCR sequencing of the targeted sequence. A homozygous RAG1-deficient rat line was generated and immunophenotyped. Meganucleases were efficient, because 3.4 and 0.6% of mouse and rat microinjected zygotes, respectively, generated mutated animals. RAG1-deficient rats showed significantly decreased proportions and numbers of immature and mature T and B lymphocytes and normal NK cells vs. littermate wild-type controls. In summary, we describe the use of engineered meganucleases to inactivate an endogenous gene with efficiencies comparable to those of ZFNs and TALENs. Moreover, we generated an immunodeficient rat line useful for studies in which there is a need for biological parameters to be analyzed in the absence of immune responses.
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Affiliation(s)
- Séverine Ménoret
- Institut National de Santé et de Recherche Médicale (INSERM) Unité Mixte de Recherche1064, Center for Research in Transplantation and Immunology and Platform Transgenic Rats Nantes Infrastructures en Biologie Sante et Agronomie, Centre National de Recherche Scientifique, Nantes, France
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26
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Zschemisch NH, Glage S, Wedekind D, Weinstein EJ, Cui X, Dorsch M, Hedrich HJ. Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat. BMC Immunol 2012; 13:60. [PMID: 23136839 PMCID: PMC3522011 DOI: 10.1186/1471-2172-13-60] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2012] [Accepted: 11/02/2012] [Indexed: 11/18/2022] Open
Abstract
Background Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain. Results After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat. Conclusion The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.
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Affiliation(s)
- Nils-Holger Zschemisch
- Institute of Laboratory Animal Science, Hannover Medical School, Carl-Neuberg-Str,1, 30625, Hannover, Germany.
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27
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28
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A retrospective analysis of germline competence in rat embryonic stem cell lines. Transgenic Res 2012; 22:411-6. [PMID: 22875289 DOI: 10.1007/s11248-012-9638-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2012] [Accepted: 07/20/2012] [Indexed: 12/23/2022]
Abstract
The factors responsible for conferring germline competence in embryonic stem (ES) cell lines remain unidentified. In the present study, rat ES cell lines (n = 17) were established with 3i medium (SU5402, PD0325901, CHIR99021), 2i medium (PD0325901, CHIR99021) or 2iF medium (PD0325901, CHIR99021, forskolin), and their potential for germline transmission to the G1 generation was examined. Rat strains were divided into an albino group (F344, Wistar or CAG/Venus transgenic rats with the Wistar background) or a colored coat group (Brown-Norway, Dark-Agouti, or BLK rats selected from >F3 generations of Wistar × Dark-Agouti rats based on their black coat color). Successful germline transmission was observed in 57 % (4/7), 40 % (2/5) and 100 % (5/5) of the ES cells established with 3i, 2i and 2iF media, respectively. ES cell lines from the homozygous CAG/Venus transgenic rats were established in all three media, but only the lines established with the 2iF medium were germline-competent. Neither coat-color (albino: 64 %, 7/11; colored: 67 %, 4/6) nor gender of the ES cell lines (XX: 67 %, 2/3; XY: 64 %, 9/14) were likely to affect germline transmission.
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Zheng S, Geghman K, Shenoy S, Li C. Retake the center stage--new development of rat genetics. J Genet Genomics 2012; 39:261-8. [PMID: 22749013 DOI: 10.1016/j.jgg.2012.05.003] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2012] [Revised: 05/02/2012] [Accepted: 05/02/2012] [Indexed: 10/28/2022]
Abstract
The rat is a powerful model for the study of human physiology and diseases, and is preferred by physiologists, neuroscientists and toxicologists. However, the lack of robust genetic modification tools has severely limited the generation of rat genetic models over the last two decades. In the last few years, several gene-targeting strategies have been developed in rats using N-ethyl-N-nitrosourea (ENU), transposons, zinc-finger nucleases (ZFNs), bacterial artificial chromosome (BAC) mediated transgenesis, and recently established rat embryonic stem (ES) cells. The development and improvement of these approaches to genetic manipulation have created a bright future for the use of genetic rat models in investigations of gene function and human diseases. Here, we summarize the strategies used for rat genetic manipulation in current research. We also discuss BAC transgenesis as a potential tool in rat transgenic models.
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Affiliation(s)
- Sushuang Zheng
- Department of Neurology, Friedman Brain Institute, Mt. Sinai School of Medicine, Box 1137, New York, NY 10029, USA
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Hirabayashi M, Tamura C, Sanbo M, Goto T, Kato-Itoh M, Kobayashi T, Nakauchi H, Hochi S. Ability of tetraploid rat blastocysts to support fetal development after complementation with embryonic stem cells. Mol Reprod Dev 2012; 79:402-12. [PMID: 22499253 DOI: 10.1002/mrd.22043] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2011] [Accepted: 03/29/2012] [Indexed: 01/20/2023]
Abstract
This study was undertaken to generate rat offspring via tetraploid blastocyst complementation with embryonic stem (ES) cells. Tetraploid blastocysts were prepared by electrofusion of blastomeres from two-cell stage embryos, and subsequent in vivo culture for 4 days. Microinjection into the tetraploid blastocoel of an inner cell mass isolated by immunosurgery resulted in the generation of rat offspring, suggesting the successful contribution of tetraploid blastocysts to their placenta. Tetraploid blastocyst complementation was attempted with a total of 4 ES cell lines (2 lines of female karyotype and 2 lines of male karyotype). In the rESWIv-3i-5 (XX) cell line, normal-sized fetuses with heartbeats were harvested on E11.5 (12.1%), E12.5 (9.5%), and E13.5 (9.1%), but no viable fetuses were detected on E14.5. Similarly, use of the rESWIv-3i-1 (XX) cell line resulted in no viable fetus production on E14.5. Using the rESBLK2i-1 (XY) cell line, viable fetuses were harvested not only on E11.5-E13.5 (2.6-5.5%), but also on E14.5 (3.0%). The transfer of a total of 487 tetraploid blastocysts complemented with rESBLK2i-1 cells resulted in 256 implantation sites (52.6%) on E21.5, but no viable offspring was detected. Use of the rESBLK2i-1/huKO (XY) cell line also resulted in no viable offspring production on E21.5. Analyses of the methylation pattern in differentially methylated regions and transcript level of genes that are imprinted in mice (H19, Meg3, Igf2r, Peg5, and Peg10) in the E14.5 conceptuses indicated a marked difference between the ES cell-derived and control normal fetuses, but not between the tetraploid and control diploid placenta.
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Affiliation(s)
- Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.
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