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
Published Date:Aug 01 2005
Source:Nucleic Acids Res. 2005; 33(13):e118.
Chromosomes, Artificial, Bacterial
Deoxyribonucleases, Type II Site-Specific
DNA Transposable Elements
Regulatory Sequences, Nucleic Acid
Funding:1P20 MD00175-01/MD/NIMHD NIH HHS/United States
1U56 CA92077-01/CA/NCI NIH HHS/United States
SO 608049/SO/PHSPO CDC HHS/United States
Description: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.
text/plain image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg text/plain
You May Also Like: