Homologous recombination in Escherichia coli simplifies the generation of gene targeting constructs for transduction into mouse embryonic stem (ES) cells1,2,3,4,5,6,7. Taking advantage of the extensive homology provided by intact bacterial artificial chromosomes (BACs), we have developed an efficient method for preparing targeted gene disruptions in ES cells. Correctly integrated clones were identified by a simple screening procedure based on chromosomal fluorescence in situ hybridization (FISH). To date, five mutant lines have been generated and bred to homozygosity by this approach.
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We thank Naifang Lu and Jeannie T. Lee for help in developing the FISH protocol, Naifang Lu for blastocyst injections, and Vidya Kunjathoor, Yanhong Ma, and Amy Stirman for assistance. This work was supported by grants from the US National Institutes of Health (AI27849, AI46731, and HL66678 to B.S.) and a postdoctoral fellowship from the Cancer Research Institute (to Y.Y.).
The authors declare no competing financial interests.
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Yang, Y., Seed, B. Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes. Nat Biotechnol 21, 447–451 (2003). https://doi.org/10.1038/nbt803
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