The rat is the preferred experimental animal in many biological studies. [1]. Its size, physiology, intelligence and reproductive characteristics make it a particularly useful model to study most facets of mammalian biology, including human disease. Despite these advantages, progress in applying forward genetic approaches to dissect the genetic and molecular basis of biological processes in rats has languished behind the rapid advances made in mice, particularly those made through applying homologous recombination in embryonic stem (ES) cells. A limiting step in applying this technology to rats has been the lack of genuine germ line qualified rat ES cells. However, a novel serum-free culture system using small molecule differentiation inhibitors was recently shown to support the derivation and propagation of genuine rat ES cell lines [2], [3]. These cell lines can be transmitted through the germ line and provide an opportunity to apply contemporary in-vivo DNA recombination based methods 1227633-49-9 supplier 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 locus. The hprt enzyme catalyses a key step in the scavenger pathway for purine synthesis and its inactivation can be selected for directly, either positively or negatively, by chemically manipulating nucleotide biosynthesis. The gene encoding HPRT is usually located on the X-chromosome and was amongst the first genes to be successfully targeted by homologous recombination in mouse, in an attempt to model the mutation that causes Lesch-Nyhan syndrome in humans [4], [5]. Manipulation of the gene also has direct applications in genetic engineering [6], [7], [8], [9]. The locus, with its ubiquitous, low level, constitutive transcriptional activity can be exploited as a safe haven for expressing exogenous transgenes [10]. Targeted integration of transgenes within the locus, using, for example, recombination mediated cassette exchange [11], [12], permits both comparative analysis of genes placed at the identical genomic site, as well as tight experimental control of conditionally regulated transgenes [13], [14], [15]. In addition, minigenes can be used in chromosome engineering [8], [9], [16]. In this report we demonstrate efficient homologous recombination at Mouse monoclonal to CD95 the locus in ES cells derived from inbred and outbred strains of rats. We compared the targeting efficiencies in these lines with those previously obtained with ES cells of other species, and evaluated the differentiation potential of correctly targeted clones, to assess the feasibility of gene targeting in the rat using ES cells. Results and Discussion Based on previous reports describing targeted disruption of the gene in mouse and human ES cells, the targeting vector was designed 1227633-49-9 supplier to delete exons 7 and 8 of the rat gene, thereby ensuring its complete inactivation (Physique 1). 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. Sub-fragments of this amplicon, flanking exons 7 1227633-49-9 supplier and 8, provided the 5 and 3 homology arms used to encompass a dual positive/unfavorable selection cassette within the targeting vector. This cassette contains a PGK-neo transcription unit to allow positive selection of G418 resistant transfectants, and a MC1-thymidine kinase (TK) minigene that enables unfavorable selection using gancyclovir, thereby facilitating substitution of the entire cassette by recombination-mediated cassette exchange via flanking heterospecific LoxP and Lox511 sites (Physique 1). Physique 1 Targeting of the gene in rat embryonic stem cells. To establish the general applicability of gene targeting in rat ES cells we decided to disrupt the gene in cell lines from two rat strains. The Fischer F344 strain was selected as representing an inbred rat that is usually frequently used.