PCR-BASED GENE TARGETING OF THE INDUCIBLE NITRIC-OXIDE SYNTHASE (NOS2) LOCUS IN MURINE ES CELLS, A NEW AND MORE COST-EFFECTIVE APPROACH

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular conseque nces of specific genetic mutations. A typical targeting construct cons ists of a neomycin phosphotransferase (neo) gene flanked by genomic DN A fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine ge nomic DNA library. Here we describe an alternative approach whereby th e inducible nitric oxide synthase (NOS2) gene locus is partially mappe d and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where th e neo gene is flanked by PCR-derived homologous DNA sequences. The vec tor also includes a thymidine kinase (tk) negative-selectable marker g ene. Following transfection into ES cells, the PCR-based targeting vec tor undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the co nventional cloning approach. It expedites the acquisition of homologou s genomic DNA sequences and simplifies the construction of targeting p lasmids by making use of defined cloning sites. This approach should r esult in substantial time and cost savings for appropriate homologous recombination projects.