Nucleic Acids Research, 2005, Vol. 33, No. 13 e118 doi:10.1093/nar/gni119 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. Shakes 1 , Douglas M. Garland1,2, Deepak K. Srivastava1, Ken R. Harewood1 and Pradeep K. Chatterjee1,* 1Julius L. Chambers Biomedical/Biotechnology Research Institute and 2Department of Biology, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA Received July 11, 2005; Revised and Accepted July 13, 2005 ABSTRACT 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 pro- cessed 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 trans- poson, should facilitate functionally mapping long- range gene regulatory sequences and help to isolate genes with defined functional boundaries in numer- ous projects including those of therapeutic interest. INTRODUCTION A comparison of genomic sequences indicates much of the DNA that is highly conserved between human and mouse do not actually code for proteins (1), but is thought to be involved in regulating gene expression (2,3). Part of this conserved non- coding DNA comprises Cis-acting sequences, sometimes loc- ated far away from the coding region of a gene, regulating transcription (2–5). Identifying these functionally presents a major challenge because traditional approaches have addressed only short range interactions between DNA sequence modules in small plasmids. Localizing regulatory sequences distal to a gene function- ally requires that the gene and its regulators be housed in large insert clones such as BACs or PACs (6–8). Libraries of clones with large inserts and high coverage of human, mouse and rat DNA exist (9–11), and the availability of a set of BAC clones spanning the entire human genome has set the stage for serious functional mapping projects (12). Procedures capable of trun- cating large genomic inserts in clones from these libraries should facilitate such effort (13–19). Methods that progressively delete DNA from one end of genomic inserts in BACs and PACs have been described, and used to localize genetic markers on a physical map of the chromosome (20,21). Cre recombination of a randomly trans- posed loxP site to insert DNA with one endogenous to the clone generates a deletion series. The resulting truncations are recovered by packaging in a phage P1, with the size of clones limited to 110 kb. Despite this size limitation, the ease of scanning 100 kb of conserved non-coding DNA for gene regu- latory sequences quickly is a distinct advantage, and the pro- cedure was used recently to functionally identify three new enhancer sequences 27 kb upstream of the Nkx2-5 gene in transgenic mice (3). *To whom correspondence should be addressed. Tel: +1 919 530 7017; Fax: +1 919 530 7998; Email: pchatterjee@nccu.edu 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@oupjournals.org