Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells

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

doi:10.1371/journal.pone.0014225

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.

Highlights

The rat was first domesticated for scientific research over 100 years ago and rapidly became one of the most important experimental animal models in biomedical sciences [1]
In this report we demonstrate efficient homologous recombination at the hprt locus in embryonic stem (ES) cells derived from inbred and outbred strains of rats
A 7 kb fragment spanning this region was amplified from Fischer 344 (F344) rat genomic DNA by PCR, using oligonucleotide primers based on genomic sequence information available for the Brown Norway (BN) strain

Summary

Introduction

The rat was first domesticated for scientific research over 100 years ago and rapidly became one of the most important experimental animal models in biomedical sciences [1]. These cell lines can be transmitted through the germ line and provide an opportunity to apply contemporary invivo DNA recombination based methods to deliver targeted genetic engineering in the rat. To evaluate the potential of these novel rat ES cell lines for introducing targeted mutations in the rat, we have tested their capacity for homologous recombination at the hprt locus.

Results

Conclusion