Explorer Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells

Content Provider	Semantic Scholar
Author	Meek, Stephen E. Buehr, Mia Sutherland, Linda Thomson, Alison Mullins, John J. Smith, Andrew J. Burdon, Tom
Copyright Year	2017
Abstract	The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat. Citation: Meek S, Buehr M, Sutherland L, Thomson A, Mullins JJ, et al. (2010) Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells. PLoS ONE 5(12): e14225. doi:10.1371/journal.pone.0014225 Editor: David S. Milstone, Brigham and Women’s Hospital, United States of America Received April 20, 2010; Accepted November 15, 2010; Published December 3, 2010 Copyright: 2010 Meek et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Biological and Biotechnological Science Research Council and the Roslin Foundation (SM, MB, LS, AT, TB). JJM was the recipient of a Wellcome Trust Principal Research Fellowship and his contribution here was supported by the EURATOOLS programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tom.burdon@roslin.ed.ac.uk
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Language	English
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