Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors.
Contrary to several earlier reports, we find that cross-recombination between wild-type and the mutant loxP511 sites is <0.5% of that between two wild-type sites if Cre protein is expressed by phage P1 during an infection. The finding enabled us to develop a procedure to truncate DNA progressively from both ends of large genomic inserts flanked by these two loxP sites in pBACe3.6 and related vectors with transposons carrying either a wild-type or a loxP511 sequence. Newly constructed loxP511 transposons contained either a kanamycin resistance gene or no marker. Insert DNA ends in deletions were sequenced with primers unique to each transposon-end remaining after the respective recombination. End-sequencing 223 deletions confirmed that the low level of cross-recombination, observed between those sites during the P1 transductions, does not complicate the procedure: truncations from the unintended end of genomic inserts did not occur. Multiple BACs pooled together could also be processed in a single tube to make end-deletions. This deletion technology, utilizing the very minimal cross-recombination between the mutant and wild-type loxP sites of most BAC clones in the public domain and a heterologous one inserted as a transposon, should facilitate functionally mapping long-range gene regulatory sequences and help to isolate genes with defined functional boundaries in numerous projects including those of therapeutic interest.
Shakes
Leighcraft A
LA
Julius L. Chambers Biomedical/Biotechnology Research Institute Durham, NC 27707, USA.
Garland
Douglas M
DM
Srivastava
Deepak K
DK
Harewood
Ken R
KR
Chatterjee
Pradeep K
PK
D017101:Bacteriophage P1 / Q000235:genetics; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D004251:DNA Transposable Elements; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019426:Integrases / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; 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; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases