A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli.
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.
Lalioti
M
M
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. m.lalioti@bham.ac.uk
Heath
J
J
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
D017468:Receptors, Fibroblast Growth Factor; D012313:RNA