Dual fluorescent reporter pig for cre recombination: Transgene placement at the ROSA26 locus

Abstract

We are extending the Cre/loxP site-specific recombination system to pigs, focussing on conditional and tissue-specific expression of oncogenic mutations to model human cancers. Identifying the location, pattern and extent of Cre recombination in vivo is an important aspect of this technology. Here we report pigs with a dual fluorochrome cassette under the control of the strong CAG promoter that switches expression after Cre-recombination, from membrane-targeted tandem dimer Tomato to membrane-targeted green fluorescent protein. The reporter cassette was placed at the porcine ROSA26 locus by conventional gene targeting using primary mesenchymal stem cells, and animals generated by nuclear transfer. Gene targeting efficiency was high, and analysis of foetal organs and primary cells indicated that the reporter is highly expressed and functional. Cre reporter pigs will provide a multipurpose indicator of Cre recombinase activity, an important new tool for the rapidly expanding field of porcine genetic modification. © 2014 Li et al.