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1
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Meek S, Singh-Dolt K, Sutherland L, Sharp MGF, Del-Pozo J, Walker D, Burdon T. Redundancy of p75NTR neurotrophin receptor function in development, growth and fertility in the rat. Transgenic Res 2024; 33:255-266. [PMID: 38981975 PMCID: PMC11319401 DOI: 10.1007/s11248-024-00395-9] [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: 04/04/2024] [Accepted: 07/01/2024] [Indexed: 07/11/2024]
Abstract
The p75NTR neurotrophin receptor has positive and negative roles regulating cell survival in the nervous system. Unambiguous interpretation of p75NTR function in vivo has been complicated, however, by residual expression of alternate forms of p75NTR protein in initial p75NTR knock-out mouse models. As rats are the preferred rodent for studying brain and behaviour, and to simplify interpretation of the knock-out phenotype, we report here the generation of a mutant rat devoid of the p75NTR protein. TALEN-mediated recombination in embryonic stem cells (ESCs) was used to flank exon 2 of p75NTR with Lox P sites and produce transgenic rats carrying either un-recombined floxed p75NTREx2-fl, or recombined, exon-2 deleted p75NTREx2-Δ alleles. Crossing p75NTREx2-fl rats with a Cre-deleter strain efficiently removed exon 2 in vivo. Excision of exon 2 causes a frameshift after p75NTR Gly23 and eliminated p75NTR protein expression. Rats lacking p75NTR were healthy, fertile, and histological analysis did not reveal significant changes in cellular density or overall structure in their brains. p75NTR function is therefore largely dispensable for normal development, growth and basal homeostasis in the rat. However, the availability of constitutive and conditional p75NTREx2-Δ rats provides new opportunities to investigate specific roles of p75NTR upon injury and during tissue repair.
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Affiliation(s)
- Stephen Meek
- The Roslin Institute, RDSVS, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK.
| | - Karamjit Singh-Dolt
- The Roslin Institute, RDSVS, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK
- Leiden University Medical Center, Leiden University, Leiden, The Netherlands
| | - Linda Sutherland
- The Roslin Institute, RDSVS, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK
| | - Matthew G F Sharp
- Bioresearch and Veterinary Services, University of Edinburgh, Chancellors Building, 49 Little France Crescent, Edinburgh, EH16 4SB, UK
| | - Jorge Del-Pozo
- The Royal Dick School of Veterinary Science, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK
| | - David Walker
- The Royal Dick School of Veterinary Science, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK
- VPG Histology, 637 Gloucester Rd, Horfield, Bristol, BS7 0BJ, UK
| | - Tom Burdon
- The Roslin Institute, RDSVS, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK.
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2
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Tian Z, Yu T, Liu J, Wang T, Higuchi A. Introduction to stem cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2023; 199:3-32. [PMID: 37678976 DOI: 10.1016/bs.pmbts.2023.02.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/17/2023]
Abstract
Stem cells have self-renewal capability and can proliferate and differentiate into a variety of functionally active cells that can serve in various tissues and organs. This review discusses the history, definition, and classification of stem cells. Human pluripotent stem cells (hPSCs) mainly include embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Embryonic stem cells are derived from the inner cell mass of the embryo. Induced pluripotent stem cells are derived from reprogramming somatic cells. Pluripotent stem cells have the ability to differentiate into cells derived from all three germ layers (endoderm, mesoderm, and ectoderm). Adult stem cells can be multipotent or unipotent and can produce tissue-specific terminally differentiated cells. Stem cells can be used in cell therapy to replace and regenerate damaged tissues or organs.
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Affiliation(s)
- Zeyu Tian
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Tao Yu
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Jun Liu
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Ting Wang
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
| | - Akon Higuchi
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China; Department of Chemical and Materials Engineering, National Central University, Jhongli, Taoyuan, Taiwan.
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3
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Sherstyuk VV, Zakian SM. Generation of Transgenic Rat Embryonic Stem Cells Using the CRISPR/Cpf1 System for Inducible Gene Knockout. BIOCHEMISTRY (MOSCOW) 2021; 86:843-851. [PMID: 34284709 DOI: 10.1134/s0006297921070051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism. To study functions of the essential genes, as well as the processes of cell differentiation, the method of induced knockout is widely used. The CreERT2/loxP system allows obtaining an inducible knockout in cells expressing tamoxifen-inducible Cre recombinase (CreERT2) and containing loxP sites flanking the target gene by adding 4-hydroxy tamoxifen to the culture medium. However, the rat ESC lines expressing CreERT2 are absent. In this work, we tested three CRISPR/Cas systems for introduction of double-strand breaks into the Rosa26 locus in the rat ESCs and inserted tamoxifen-dependent Cre recombinase into this locus using the CRISPR/Cpf1 system. It was shown that the obtained transgenic rat ESC lines retained the characteristics of pluripotent cells. Tamoxifen-inducible Cre recombinase activity was analyzed using a reporter vector.
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Affiliation(s)
- Vladimir V Sherstyuk
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
| | - Suren M Zakian
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia
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Szpila M, Humięcka M, Bożyk K, Paterczyk B, Suwińska A, Maleszewski M, Tarkowski AK. Attempts to obtain fully xenogeneic fetuses in rat ↔ mouse model†,‡. Biol Reprod 2021; 102:499-510. [PMID: 31511860 DOI: 10.1093/biolre/ioz185] [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: 04/10/2019] [Revised: 08/06/2019] [Accepted: 09/06/2019] [Indexed: 11/15/2022] Open
Abstract
The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse ↔ rat chimeras, the interspecific uterine barrier is less hostile to interspecific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse ↔ rat blastocysts by injection of mouse embryonic stem cells (ESCs) into eight-cell rat embryos and rat ESCs into eight-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found that the xenogeneic fetuses are eliminated at the perigastrulation stage of development. Thus, we conclude that in the rat ↔ mouse combination even when extraembryonic tissues of the chimeric embryo are composed solely of the cells of the same species as the female to which embryos are transferred, the full-term development of the pure xenogeneic fetus is very unlikely.
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Affiliation(s)
- Marcin Szpila
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Monika Humięcka
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Katarzyna Bożyk
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Bohdan Paterczyk
- Laboratory of Electron and Confocal Microscopy, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Aneta Suwińska
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Marek Maleszewski
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Andrzej K Tarkowski
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
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5
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Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats. Nat Commun 2021; 12:1328. [PMID: 33637711 PMCID: PMC7910474 DOI: 10.1038/s41467-021-21557-x] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Accepted: 01/29/2021] [Indexed: 02/06/2023] Open
Abstract
Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.
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6
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Borkowska M, Leitch HG. Mouse Primordial Germ Cells: In Vitro Culture and Conversion to Pluripotent Stem Cell Lines. Methods Mol Biol 2021; 2214:59-73. [PMID: 32944903 DOI: 10.1007/978-1-0716-0958-3_5] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
Primordial germ cells (PGCs) are the embryonic precursors of the gametes. Despite decades of research, in vitro culture of PGCs remains a major challenge and has previously relied on undefined components such as serum and feeders. Notably, PGCs cultured for extended periods do not maintain their lineage identity but instead undergo conversion to form pluripotent stem cell lines called embryonic germ (EG) cells in response to LIF/STAT3 signaling. Here we report both established and new methodologies to derive EG cells, in a range of different conditions. We show that basic fibroblast growth factor is not required for EG cell conversion. We detail the steps taken in our laboratory to systematically remove complex components and establish a fully defined protocol that allows efficient conversion of isolated PGCs to pluripotent EG cells. In addition, we demonstrate that PGCs can adhere and proliferate in culture without the support of feeder cells or serum. This may well suggest novel approaches to establishing short-term culture of PGCs in defined conditions.
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Affiliation(s)
- Malgorzata Borkowska
- MRC London Institute of Medical Sciences (LMS), London, UK
- Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK
| | - Harry G Leitch
- MRC London Institute of Medical Sciences (LMS), London, UK.
- Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK.
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7
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Comparative Metabolomic Profiling of Rat Embryonic and Induced Pluripotent Stem Cells. Stem Cell Rev Rep 2020; 16:1256-1265. [DOI: 10.1007/s12015-020-10052-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/28/2020] [Indexed: 02/06/2023]
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8
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Meek S, Wei J, Oh T, Watson T, Olavarrieta J, Sutherland L, Carlson DF, Salzano A, Chandra T, Joshi A, Burdon T. A Stem Cell Reporter for Investigating Pluripotency and Self-Renewal in the Rat. Stem Cell Reports 2020; 14:154-166. [PMID: 31902707 PMCID: PMC6962659 DOI: 10.1016/j.stemcr.2019.12.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2019] [Revised: 11/29/2019] [Accepted: 12/02/2019] [Indexed: 12/02/2022] Open
Abstract
Rat embryonic stem cells (rESCs) are capable of contributing to all differentiated tissues, including the germ line in chimeric animals, and represent a unique, authentic alternative to mouse embryonic stem cells for studying stem cell pluripotency and self-renewal. Here, we describe an EGFP reporter transgene that tracks expression of the benchmark naive pluripotency marker gene Rex1 (Zfp42) in the rat. Insertion of the EGFP reporter gene downstream of the Rex1 promoter disrupted Rex1 expression, but REX1-deficient rESCs and rats were viable and apparently normal, validating this targeted knockin transgene as a neutral reporter. The Rex1-EGFP gene responded to self-renewal/differentiation factors and validated the critical role of β-catenin/LEF1 signaling. The stem cell reporter also allowed the identification of functionally distinct sub-populations of cells within rESC cultures, thus demonstrating its utility in discriminating between cell states in rat stem cell cultures, as well as providing a tool for tracking Rex1 expression in the rat.
Rex1-EGFP transgene is a neutral reporter of pluripotency and self-renewal in the rat Rex1-EGFP transgene responds appropriately to self-renewal and differentiation signaling Rex1-EGFP transgene allows the discrimination between rat ESC pluripotent states
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Affiliation(s)
- Stephen Meek
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
| | - Jun Wei
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK; iRegene Therapeutics, C6-522, 666 Gaoxin Avenue, Wuhan, 430070, China
| | - Taeho Oh
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
| | - Tom Watson
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
| | - Jaime Olavarrieta
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
| | - Linda Sutherland
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
| | - Daniel F Carlson
- Recombinetics Inc., 1246 University Avenue W, St. Paul, MN 55125, USA
| | - Angela Salzano
- MRC Unit for Human Genetics, Institute of Genetics and Molecular Medicine, The University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU, UK
| | - Tamir Chandra
- MRC Unit for Human Genetics, Institute of Genetics and Molecular Medicine, The University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU, UK
| | - Anagha Joshi
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
| | - Tom Burdon
- The Roslin Institute and R(D)VS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK.
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9
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Pridans C, Raper A, Davis GM, Alves J, Sauter KA, Lefevre L, Regan T, Meek S, Sutherland L, Thomson AJ, Clohisey S, Bush SJ, Rojo R, Lisowski ZM, Wallace R, Grabert K, Upton KR, Tsai YT, Brown D, Smith LB, Summers KM, Mabbott NA, Piccardo P, Cheeseman MT, Burdon T, Hume DA. Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus. THE JOURNAL OF IMMUNOLOGY 2018; 201:2683-2699. [PMID: 30249809 PMCID: PMC6196293 DOI: 10.4049/jimmunol.1701783] [Citation(s) in RCA: 104] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/29/2017] [Accepted: 08/20/2018] [Indexed: 12/23/2022]
Abstract
We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.
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Affiliation(s)
- Clare Pridans
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom; .,The University of Edinburgh Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom
| | - Anna Raper
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Gemma M Davis
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Joana Alves
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Kristin A Sauter
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Lucas Lefevre
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Tim Regan
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Stephen Meek
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Linda Sutherland
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Alison J Thomson
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom.,New World Laboratories, Laval, Quebec H7V 5B7, Canada
| | - Sara Clohisey
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Stephen J Bush
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom.,Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom
| | - Rocío Rojo
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Zofia M Lisowski
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Robert Wallace
- Department of Orthopaedic Surgery, The University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom
| | - Kathleen Grabert
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Kyle R Upton
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom.,School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia
| | - Yi Ting Tsai
- Medical Research Council Centre for Reproductive Health, The University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom
| | - Deborah Brown
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Lee B Smith
- Medical Research Council Centre for Reproductive Health, The University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom.,Faculty of Science, University of Newcastle, Callaghan, New South Wales 2309, Australia; and
| | - Kim M Summers
- Mater Research-University of Queensland, Brisbane, Queensland 4101, Australia
| | - Neil A Mabbott
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Pedro Piccardo
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Michael T Cheeseman
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - Tom Burdon
- The Roslin Institute, The University of Edinburgh, Easter Bush EH25 9RG, United Kingdom
| | - David A Hume
- The University of Edinburgh Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom; .,Mater Research-University of Queensland, Brisbane, Queensland 4101, Australia
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10
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Chen Y, Spitzer S, Agathou S, Karadottir RT, Smith A. Gene Editing in Rat Embryonic Stem Cells to Produce In Vitro Models and In Vivo Reporters. Stem Cell Reports 2018; 9:1262-1274. [PMID: 29020614 PMCID: PMC5639479 DOI: 10.1016/j.stemcr.2017.09.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2017] [Revised: 09/05/2017] [Accepted: 09/06/2017] [Indexed: 12/13/2022] Open
Abstract
Rat embryonic stem cells (ESCs) offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here, we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions in Lef1. Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knockin of dsRed at the Sox10 locus. Two clones produced germline chimeras. Comparative expression of dsRed and SOX10 validated the fidelity of the reporter. To illustrate utility, live imaging of dsRed in neonatal brain slices was employed to visualize oligodendrocyte lineage cells for patch-clamp recording. Overall, these results show that CRISPR/Cas9 gene editing technology in germline-competent rat ESCs is enabling for in vitro studies and for generating genetically modified rats.
Gene mutation and homologous recombination in rat ESCs using CRISPR/Cas9 Lef1 mutants exhibit predicted loss of hypersensitivity to GSK3 inhibition Sox10 knockin rat provides a vital reporter of neural crest and oligodendroglia Sox10::dsRed facilitates patch-clamp recording from oligodendroglial lineage cells
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Affiliation(s)
- Yaoyao Chen
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Sonia Spitzer
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK
| | - Sylvia Agathou
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK
| | - Ragnhildur Thora Karadottir
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK
| | - Austin Smith
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
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11
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Dahlmann J, Awad G, Dolny C, Weinert S, Richter K, Fischer KD, Munsch T, Leßmann V, Volleth M, Zenker M, Chen Y, Merkl C, Schnieke A, Baraki H, Kutschka I, Kensah G. Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture. PLoS One 2018. [PMID: 29513687 PMCID: PMC5841662 DOI: 10.1371/journal.pone.0192652] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.
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Affiliation(s)
- Julia Dahlmann
- Clinic of Cardiothoracic Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - George Awad
- Clinic of Cardiothoracic Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Carsten Dolny
- Clinic of Cardiothoracic Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Sönke Weinert
- Clinic of Cardiology and Angiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Karin Richter
- Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Klaus-Dieter Fischer
- Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Thomas Munsch
- Institute of Physiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Volkmar Leßmann
- Institute of Physiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Marianne Volleth
- Institute of Human Genetics, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Martin Zenker
- Institute of Human Genetics, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Yaoyao Chen
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom
| | - Claudia Merkl
- Chair of Livestock Biotechnology, Technical University Munich, Freising-Weihenstephan, Germany
| | - Angelika Schnieke
- Chair of Livestock Biotechnology, Technical University Munich, Freising-Weihenstephan, Germany
| | - Hassina Baraki
- Clinic of Cardiothoracic Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Ingo Kutschka
- Clinic of Cardiothoracic Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - George Kensah
- Clinic of Cardiothoracic Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
- * E-mail:
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12
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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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13
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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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14
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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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15
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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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16
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Li M, Li L, Zhang J, Verma V, Liu Q, Shi D, Huang B. An Insight on Small Molecule Induced Foot-Print Free Naive Pluripotent Stem Cells in Livestock. ACTA ACUST UNITED AC 2015. [DOI: 10.4236/scd.2015.51001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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17
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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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18
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Abstract
The ability of stem cells to self-renew and generate different lineages during development and organogenesis is a fundamental, tightly controlled, and generally unidirectional process, whereas the 'immortality' of cancer cells could be regarded as pathological self-renewal. The molecular mechanisms that underpin the generation of induced pluripotent stem cells are remarkably similar to those that are deregulated in cancer - so much so that aberrant reprogramming is tumorigenic. The similarities also suggest that mutations in genes implicated in DNA methylation dynamics might represent a hallmark of cancers with a stem cell origin, and they highlight an alternative view of cancer that may be of clinical benefit.
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Affiliation(s)
- Colin R Goding
- Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, Old Road Campus Research Building, Old Road Campus, University of Oxford, Headington, Oxford OX3 7DQ, UK
| | - Duanqing Pei
- South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530 China
| | - Xin Lu
- Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, Old Road Campus Research Building, Old Road Campus, University of Oxford, Headington, Oxford OX3 7DQ, UK
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19
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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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20
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Leitch HG, Tang WWC, Surani MA. Primordial germ-cell development and epigenetic reprogramming in mammals. Curr Top Dev Biol 2014; 104:149-87. [PMID: 23587241 DOI: 10.1016/b978-0-12-416027-9.00005-x] [Citation(s) in RCA: 94] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Primordial germ cells (PGCs) are the embryonic precursors of the gametes and represent the founder cells of the germline. Specification of PGCs is a critical divergent point during embryogenesis. Whereas the somatic lineages will ultimately perish, cells of the germline have the potential to form a new individual and hence progress to the next generation. It is therefore critical that the genome emerges intact and carrying the appropriate epigenetic information during its passage through the germline. To ensure this fidelity of transmission, PGC development encompasses extensive epigenetic reprogramming. The low cell numbers and relative inaccessibility of PGCs present a challenge to those seeking mechanistic understanding of the crucial developmental and epigenetic processes in this most fascinating of lineages. Here, we present an overview of PGC development in the mouse and compare this with the limited information available for other mammalian species. We believe that a comparative approach will be increasingly important to uncover the extent to which mechanisms are conserved and reveal the critical steps during PGC development in humans.
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Affiliation(s)
- Harry G Leitch
- Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, United Kingdom
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21
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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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22
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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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23
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Robust self-renewal of rat embryonic stem cells requires fine-tuning of glycogen synthase kinase-3 inhibition. Stem Cell Reports 2013; 1:209-17. [PMID: 24319657 PMCID: PMC3849254 DOI: 10.1016/j.stemcr.2013.07.003] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2013] [Revised: 07/16/2013] [Accepted: 07/17/2013] [Indexed: 02/02/2023] Open
Abstract
Germline-competent embryonic stem cells (ESCs) have been derived from mice and rats using culture conditions that include an inhibitor of glycogen synthase kinase 3 (GSK3). However, rat ESCs remain susceptible to sporadic differentiation. Here, we show that unsolicited differentiation is attributable to overinhibition of GSK3. The self-renewal effect of inhibiting GSK3 is mediated via β-catenin, which abrogates the repressive action of TCF3 on core pluripotency genes. In rat ESCs, however, GSK3 inhibition also leads to activation of differentiation-associated genes, notably lineage specification factors Cdx2 and T. Lowered GSK3 inhibition reduces differentiation and enhances clonogenicity and self-renewal. The differential sensitivity of rat ESCs to GSK3 inhibition is linked to elevated expression of the canonical Wnt pathway effector LEF1. These findings reveal that optimal GSK3 inhibition for ESC propagation is influenced by the balance of TCF/LEF factors and can vary between species.
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24
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Leitch H, Nichols J, Humphreys P, Mulas C, Martello G, Lee C, Jones K, Surani M, Smith A. Rebuilding pluripotency from primordial germ cells. Stem Cell Reports 2013; 1:66-78. [PMID: 24052943 PMCID: PMC3757743 DOI: 10.1016/j.stemcr.2013.03.004] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2013] [Revised: 03/22/2013] [Accepted: 03/23/2013] [Indexed: 02/02/2023] Open
Abstract
Mammalian primordial germ cells (PGCs) are unipotent progenitors of the gametes. Nonetheless, they can give rise directly to pluripotent stem cells in vitro or during teratocarcinogenesis. This conversion is inconsistent, however, and has been difficult to study. Here, we delineate requirements for efficient resetting of pluripotency in culture. We demonstrate that in defined conditions, routinely 20% of PGCs become EG cells. Conversion can occur from the earliest specified PGCs. The entire process can be tracked from single cells. It is driven by leukemia inhibitory factor (LIF) and the downstream transcription factor STAT3. In contrast, LIF signaling is not required during germ cell ontogeny. We surmise that ectopic LIF/STAT3 stimulation reconstructs latent pluripotency and self-renewal. Notably, STAT3 targets are significantly upregulated in germ cell tumors, suggesting that dysregulation of this pathway may underlie teratocarcinogenesis. These findings demonstrate that EG cell formation is a robust experimental system for exploring mechanisms involved in reprogramming and cancer.
A defined system for generation of pluripotent EG cells at high efficiency 20% of single primordial germ cells become EG cells Stimulation with LIF but not FGF drives conversion to pluripotency LIF/STAT3 targets are upregulated in pluripotent germ cell tumors
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Affiliation(s)
- Harry G. Leitch
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Jennifer Nichols
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Department of Physiology, Development, and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Peter Humphreys
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Carla Mulas
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Graziano Martello
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Caroline Lee
- Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Ken Jones
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - M. Azim Surani
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Department of Physiology, Development, and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Austin Smith
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Corresponding author
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25
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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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26
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Insertional mutagenesis by a hybrid piggyBac and sleeping beauty transposon in the rat. Genetics 2012; 192:1235-48. [PMID: 23023007 DOI: 10.1534/genetics.112.140855] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.
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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: 13] [Impact Index Per Article: 1.0] [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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Abstract
Specific cells within the early mammalian embryo have the capacity to generate all somatic lineages plus the germline. This property of pluripotency is confined to the epiblast, a transient tissue that persists for only a few days. In vitro, however, pluripotency can be maintained indefinitely through derivation of stem cell lines. Pluripotent stem cells established from the newly formed epiblast are known as embryonic stem cells (ESCs), whereas those generated from later stages are called postimplantation epiblast stem cells (EpiSCs). These different classes of pluripotent stem cell have distinct culture requirements and gene expression programs, likely reflecting the dynamic development of the epiblast in the embryo. In this chapter we review current understanding of how the epiblast forms and relate this to the properties of derivative stem cells. We discuss whether ESCs and EpiSCs are true counterparts of different phases of epiblast development or are culture-generated phenomena. We also consider the proposition that early epiblast cells and ESCs may represent a naïve ground state without any prespecification of lineage choice, whereas later epiblasts and EpiSCs may be primed in favor of particular fates.
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Affiliation(s)
- Jennifer Nichols
- Wellcome Trust Centre for Stem Cell Research, Stem Cell Institute University of Cambridge, Cambridge CB2 1QR, United Kingdom
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