Selection-marker-free modification of the murine beta-casein gene using a lox2272 [correction of lox2722] site - PubMed (original) (raw)

. 2001 Mar;290(2):260-71.

doi: 10.1006/abio.2000.4984.

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Selection-marker-free modification of the murine beta-casein gene using a lox2272 [correction of lox2722] site

A F Kolb. Anal Biochem. 2001 Mar.

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Abstract

Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site. DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the beta-casein gene in murine embryonic stem cells. The incompatibility of the heterospecific lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification. This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene.

Copyright 2001 Academic Press.

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