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dc.contributor.authorChen, Yen
dc.contributor.authorSpitzer, Sen
dc.contributor.authorAgathou, Sen
dc.contributor.authorKaradottir, Thoraen
dc.contributor.authorSmith, Austinen
dc.date.accessioned2017-11-08T14:06:36Z
dc.date.available2017-11-08T14:06:36Z
dc.date.issued2017-10-10en
dc.identifier.issn2213-6711
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/268180
dc.description.abstractRat 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.
dc.description.sponsorshipThis 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).
dc.publisherElsevier
dc.rightsAttribution 4.0 International*
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectgene targetingen
dc.subjectCRISPR/Cas9en
dc.subjectoligodendrocyteen
dc.subjectneural cresten
dc.subjectraten
dc.subjecttransgenesisen
dc.subjecthomologous recombinationen
dc.subjectpluripotencyen
dc.subjectSOX10en
dc.subjectembryonic stem cellsen
dc.titleGene Editing in Rat Embryonic Stem Cells to Produce In Vitro Models and In Vivo Reportersen
dc.typeArticle
prism.endingPage1274
prism.issueIdentifier4en
prism.publicationDate2017en
prism.publicationNameStem Cell Reportsen
prism.startingPage1262
prism.volume9en
dc.identifier.doi10.17863/CAM.14380
dcterms.dateAccepted2017-09-06en
rioxxterms.versionofrecord10.1016/j.stemcr.2017.09.005en
rioxxterms.versionVoR*
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/en
rioxxterms.licenseref.startdate2017-10-10en
dc.contributor.orcidKaradottir, Thora [0000-0001-9675-2722]
dc.contributor.orcidSmith, Austin [0000-0002-3029-4682]
dc.identifier.eissn2213-6711
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idSwiss National Science Foundation (SNSF) (via Biozentrum University of Basel) (130441)
pubs.funder-project-idBBSRC (BB/H012737/1)
pubs.funder-project-idWellcome Trust (091543/Z/10/Z)
cam.issuedOnline2017-10-10en


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International