Gene Editing in Rat Embryonic Stem Cells to Produce In Vitro Models and In Vivo Reporters
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Publication Date
2017-10-10Journal Title
Stem Cell Reports
ISSN
2213-6711
Publisher
Elsevier
Volume
9
Issue
4
Pages
1262-1274
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Chen, Y., Spitzer, S., Agathou, S., Karadottir, T., & Smith, A. (2017). Gene Editing in Rat Embryonic Stem Cells to Produce In Vitro Models and In Vivo Reporters. Stem Cell Reports, 9 (4), 1262-1274. https://doi.org/10.1016/j.stemcr.2017.09.005
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.
Keywords
gene targeting, CRISPR/Cas9, oligodendrocyte, neural crest, rat, transgenesis, homologous recombination, pluripotency, SOX10, embryonic stem cells
Sponsorship
This research was funded by the European Community project EURATRANS (grant no. HEALTH-F4-2010-241504), the Biotechnology and Biological Sciences Research Council of the United Kingdom (grant no. BB/H012737/1), the Swiss National Science Foundation Sinergia Program, the Louis-Jeantet Foundation, and the Isaac Newton Trust. R.T.K. was supported by a Wellcome Trust Research Career Development Fellowship (grant no. 091543/Z/10/Z) and a Lister Institute Research Prize. A.S. is a Medical Research Council Professor (grant no. G1100526/1).
Funder references
Swiss National Science Foundation (SNSF) (via Biozentrum University of Basel) (130441)
BBSRC (BB/H012737/1)
Wellcome Trust (091543/Z/10/Z)
Identifiers
External DOI: https://doi.org/10.1016/j.stemcr.2017.09.005
This record's URL: https://www.repository.cam.ac.uk/handle/1810/268180
Rights
Attribution 4.0 International, Attribution 4.0 International, Attribution 4.0 International
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