Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site.
A general approach for isolating large nested deletions in P1 artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) by retrofitting with a loxP site-containing Tn10 mini-transposon is described. Cre-mediated recombination between the loxP site existing in these clones and one introduced by transposition leads to deletions and inversions of the DNA between these sites. Large deletions are selectively recovered by transducing the retrofitted PAC or BAC clones with P1 phage. The requirement that both loxP sites in the cointegrate be packaged into a P1 head ensures that only large deletions are rescued. PCR analyses identified these deletions as products of legitimate recombination between loxP sites mediated by Cre protein. BACs produce deletions much more efficiently than PACs although the former cannot be induced to greater than unit copy in cells. Mammalian cell-responsive antibiotic resistance markers are introduced as part of the transposon into genomic clone deletions for subsequent functional analysis. Most importantly, the loxP site retrofitting and P1 transduction can be performed in the same bacterial host containing these clones directly isolated from PAC or BAC libraries. These procedures should facilitate physical and functional mapping of genes and regulatory elements in these large plasmids.
Chatterjee
P K
PK
Department of Medicine, SUNY Health Science Center, 750 East Adams Street, Syracuse, NY 13210, USA. chatterp@vax.cs.hscsyr.edu
Coren
J S
JS
D017101:Bacteriophage P1 / Q000235:genetics*; D002384:Catalysis; D002702:Chloramphenicol Resistance / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000737:chemistry*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D017353:Gene Deletion; D019426:Integrases / Q000378:metabolism*; D007613:Kanamycin Resistance / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011691:Puromycin / Q000494:pharmacology; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014764:Viral Proteins
D004251:DNA Transposable Elements; D004279:DNA, Viral; D014764:Viral Proteins; D011691:Puromycin; C045073:Cre recombinase; D019426:Integrases; D009355:Neomycin