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For the success of directed mutagenesis experiments using homologous recombination in embryonic stem (ES) cells, it is essential that ES cells in culture retain the ability to populate the mouse germline. Culturing the cells in such a way as to maintain this ability presents a technical challenge. Several different methodologies have been employed but one that is commonly used is to culture the ES cells on a feeder layer of mouse primary embryonic fibroblasts (MEFs) (1,2). The gene targeting technology also requires that ES cells in culture undergo drug selection to identify the cells that integrate the targeting construct Neomycin-resistance (neo*) is the most commonly used dominant selectable gene for such experiments. To maintain a viable MEF feeder layer during drug selection it is necessary to prepare this layer with drug-resistant cells. Such neomycin-resistant feeder layers are conveniently prepared from transgenic mice expressing the neomycin-resistance gene. To increase the flexibility of the targeting technology, a second dominant selectable marker, the hygromycin-resistance gene {hyg*), has been employed. Hygromycin Bis an aminocyclitol antibiotic that acts by interfering with the ribosomal translocation and aminoacyl tRNA recognition leading to mistranslations (3). The hygromycin B resistance gene is a phosphotransferase that renders the drug inactive by adding a phosphate to position 4 (4,5). With the availability of this second selectable marker, one approach for the inactivation of both alleles of a gene has been to target the first allele with neo and the second with hyg (6,10). Additionally, when homozygous targeted mutations of closely linked genes are desired in one animal, crossing the individually targeted animals together to obtain recombinants becomes impractical. Instead, sequential targeting of the two linked genes with two selectable markers becomes the method of choice.

Transgenic mice for the preparation of hygromycin-resistant primary embryonic fibroblast feeder layers for embryonic stem cell selections