title: BAC modification through serial or simultaneous use of CRE/Lox technology.
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.
authors.affiliation: Krumlauf Laboratory, Stowers Institute for Medical Research, 1000 50th Street, Kansas City, MO 64110, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Parrish
authors.forename: Jay
authors.initials: J
authors.lastname: Unruh
authors.forename: Robb
authors.initials: R
authors.lastname: Krumlauf
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D014644:Genetic Variation; D019426:Integrases / Q000235:genetics*; D055029:Inverted Repeat Sequences; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D013050:Spectrometry, Fluorescence
chemical_list: D008164:Luminescent Proteins; C045073:Cre recombinase; D019426:Integrases