Gene Editing in Rat Embryonic Stem Cells to Produce In Vitro Models and In Vivo Reporters
Stem Cell Reports
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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
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 targeting, CRISPR/Cas9, oligodendrocyte, neural crest, rat, transgenesis, homologous recombination, pluripotency, SOX10, embryonic stem cells
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).
Swiss National Science Foundation (SNSF) (via Biozentrum University of Basel) (130441)
Wellcome Trust (091543/Z/10/Z)
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
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