Genomic targeting of a bicistronic DNA fragment by Cre-mediated site-specific recombination.
The Cre-recombinase of bacteriophage P1 catalyses site-specific recombination between DNA fragments containing loxP sites. Targeting of predefined genomic loci can be achieved by Cre-mediated linkage of a promoterless resistance marker gene to a floxed promoter pre-existing in the genome. In order to avoid the introduction of plasmid sequences into the host genome, we have constructed a series of plasmids in which the DNA segment to be integrated is flanked by two loxP sites. We show here that this floxed targeting fragment is reliably and effectively separated from the vector backbone and integrated into genomic loxP sites by Cre-mediated site-specific recombination in mammalian cells. We also demonstrate that by using this approach two convergent, promoterless coding regions can simultaneously be linked to two independent promoter elements at a pre-existing genomic loxP site. This methodology will be particularly useful for genomic targeting experiments in transgenic animals.
Kolb
A F
AF
Institute of Virology and Immunology, University of Würzburg, Germany. kolba@main.hri.sari.ac.uk
Siddell
S G
SG
D000818:Animals; D030801:Animals, Genetically Modified; D017101:Bacteriophage P1 / Q000201:enzymology*; D015139:Blotting, Southern; D002415:Cats; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter / Q000235:genetics; D019426:Integrases / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection / Q000235:genetics; D014764:Viral Proteins
D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases