Protocol

Cre Recombinase Gene Transfer In Vitro and Detection of loxP-Dependent Recombination

This protocol was adapted from "Conditional Mutagenesis of the Genome Using Site-Specific DNA Recombination," Chapter 60, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

Altering the genome of intact cells and organisms by site-specific DNA recombination has become an important gene-transfer methodology. The inclusion of exogenous recombinase target sequences within transferred DNA segments allows subsequent modifications to previously altered genomic structure that increase the utility of gene transfer and enhance experimental design. In this protocol, correctly targeted mouse embryonic stem (ES) cell clones bearing the F[tkneo] allele (containing several loxP sites) are subjected to in vitro Cre gene transfer to generate ES cell subclones bearing either Type 1 () or Type 2 (F) alleles. Type 2 ES cells are used to generate chimeric mice that are then crossed to germ-line Cre-expressing mice, such as ZP3-Cre transgenic mates. The additional time needed to breed the mice (~2-3 mo) is typically less troublesome than the cost and effort of maintaining multiple clone-derived lines of mice.