Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells

Stephen Meek, Mia Buehr, Linda Sutherland, Alison Thomson, John J. Mullins, Andrew Smith, Tom Burdon

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article numbere14225
Pages (from-to)-
Number of pages6
JournalPLoS ONE
Volume5
Issue number12
DOIs
Publication statusPublished - 3 Dec 2010

Keywords

  • Animals
  • Animals, Genetically Modified
  • Blastocyst
  • Cells, Cultured
  • Embryonic Stem Cells
  • Gene Targeting
  • Genetic Techniques
  • Hypoxanthine Phosphoribosyltransferase
  • Male
  • Models, Genetic
  • Rats
  • Rats, Inbred F344
  • Rats, Sprague-Dawley
  • Recombination, Genetic
  • Species Specificity

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