Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes.
Homologous recombination in Escherichia coli simplifies the generation of gene targeting constructs for transduction into mouse embryonic stem (ES) cells. 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.
Yang
Yi
Y
Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
Seed
Brian
B
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic / Q000235:genetics; D013234:Stem Cells / Q000502:physiology; D014161:Transduction, Genetic / Q000379:methods*