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Journal of Biomedicine and Biotechnology
Volume 2011, Article ID 924068, 12 pages
http://dx.doi.org/10.1155/2011/924068
Research Article

BAC Modification through Serial or Simultaneous Use of CRE/Lox Technology

1Krumlauf Laboratory, Stowers Institute for Medical Research, 1000 50th Street, Kansas City, MO, 64110, USA
2Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS, 66160, USA

Received 24 July 2010; Accepted 6 October 2010

Academic Editor: James Birchler

Copyright © 2011 Mark Parrish et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Bacterial Artificial Chromosomes (BACs) are vital tools in mouse genomic analyses because of their ability to propagate large inserts. The size of these constructs, however, prevents the use of conventional molecular biology techniques for modification and manipulation. Techniques such as recombineering and Cre/Lox methodologies have thus become heavily relied upon for such purposes. In this work, we investigate the applicability of Lox variant sites for serial and/or simultaneous manipulations of BACs. We show that Lox spacer mutants are very specific, and inverted repeat variants reduce Lox reaction rates through reducing the affinity of Cre for the site, while retaining some functionality. Employing these methods, we produced serial modifications encompassing four independent changes which generated a mouse HoxB BAC with fluorescent reporter proteins inserted into four adjacent Hox genes. We also generated specific, simultaneous deletions using combinations of spacer variants and inverted repeat variants. These techniques will facilitate BAC manipulations and open a new repertoire of methods for BAC and genome manipulation.