title: A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli.
abstract: Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), which contain large fragments of genomic DNA, have been successfully used as transgenes to create mouse models of dose-dependent diseases. They are also potentially valuable as transgenes for dominant diseases given that point mutations and/or small rearrangements can be accurately introduced. Here, we describe a new method to introduce small alterations in BACs, which results in the generation of point mutations with high frequency. The method involves homologous recombination between the original BAC and a shuttle vector providing the mutation. Each recombination step is monitored using positive and negative selection markers, which are the Kanamycin-resistance gene, the sacB gene and temperature-sensitive replication, all conferred by the shuttle plasmid. We have used this method to introduce four different point mutations and the insertion of the ss-galactosidase gene in a BAC, which has subsequently been used for transgenic animal production.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. m.lalioti@bham.ac.uk
authors.forename: M
authors.initials: M
authors.lastname: Lalioti
authors.forename: J
authors.initials: J
authors.lastname: Heath
mesh_terms: D000818:Animals; D001847:Bone Diseases / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004622:Embryo, Mammalian / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D017354:Point Mutation; D012313:RNA / Q000235:genetics / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes / Q000235:genetics
chemical_list: D017468:Receptors, Fibroblast Growth Factor; D012313:RNA