Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs.
Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined as efficiently as wild-type. The highly specific and efficient recombination of mutant loxP sites in vivo helped in developing a procedure to progressively truncate DNA from either end of large genomic inserts in P1-derived artificial chromosomes (PACs) using transposons that carry either a wild-type or mutant loxP sequence. PAC libraries of human DNA were constructed with inserts flanked by a wild-type and one of the two mutant loxP sites, and deletions from both ends generated in clones using newly constructed wild-type and mutant loxP transposons. Analysis of the results provides new insight into the very large co-integrates formed during P1 transduction of plasmids with loxP sites: a model with tri- and possibly multimeric co-integrates comprising the PAC plasmid, phage DNA, and transposon plasmid(s) as intermediates in the cell appears best to fit the data. The ability to truncate a large piece of DNA from both ends is likely to facilitate functionally mapping gene boundaries more efficiently, and make available precisely trimmed genes in their chromosomal contexts for therapeutic applications.
Chatterjee
Pradeep K
PK
Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA. pchatterjee@nccu.edu
Shakes
Leighcraft A
LA
Srivastava
Deepak K
DK
Garland
Douglas M
DM
Harewood
Ken R
KR
Moore
Kyle J
KJ
Coren
Jonathon S
JS
D017101:Bacteriophage P1 / Q000235:genetics; D001483:Base Sequence; D022201:Chromosomes, Artificial; D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D004251:DNA Transposable Elements / Q000235:genetics*; D016678:Genome; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; D004251:DNA Transposable Elements; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases