Genome Modification by Inclusion of loxP Transgene Sequences
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
We describe here a procedure for introducing loxP sites into the mammalian genome. A typical gene-targeting approach to create a conditional null allele is presented, in which the initial placement of loxP sites is not deleterious to allele function. This can be modified to include knock-ins of point mutations, with such mutations flanked by loxP sites that can then be recombined by Cre expression. The choice of sequence or regulatory element to modify is dependent on the experimental design. Consideration must be given to the possible production of truncated and altered gene sequence products, or otherwise aberrantly functioning alleles.
