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Nucleic Acids Research 2005 33(13):e118; doi:10.1093/nar/gni119
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Published online 1 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

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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

Leighcraft A. Shakes1, Douglas M. Garland1,2, Deepak K. Srivastava1, Ken R. Harewood1 and Pradeep K. Chatterjee1,*

1Julius L. Chambers Biomedical/Biotechnology Research Institute Durham, NC 27707, USA 2Department of Biology, North Carolina Central University 1801 Fayetteville Street, Durham, NC 27707, USA

*To whom correspondence should be addressed. Tel: +1 919 530 7017; Fax: +1 919 530 7998; Email: pchatterjee{at}nccu.edu

Received July 11, 2005. Revised July 13, 2005. Accepted July 13, 2005.

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.


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L. A. Shakes, T. L. Malcolm, K. L. Allen, S. De, K. R. Harewood, and P. K. Chatterjee
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[Abstract] [Full Text] [PDF]


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