Dual fluorescent reporter pig for Cre recombination: transgene placement at the ROSA26 locus

PLoS One. 2014 Jul 15;9(7):e102455. doi: 10.1371/journal.pone.0102455. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Genes, Reporter
  • Genetic Engineering
  • Genetic Loci
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Integrases / physiology*
  • Mesenchymal Stem Cells / physiology
  • RNA, Untranslated / genetics*
  • Sus scrofa / genetics*
  • Transgenes

Substances

  • RNA, Untranslated
  • Green Fluorescent Proteins
  • Cre recombinase
  • Integrases

Grants and funding

This work was supported financially by the Deutsche Forschungsgemeinschaft (German Research Foundation) through grant number SCHN971/3-1, WO 685/16-1. Shun Li would like to thank the China Scholarship Council for financial support for his PhD study (No. 2011699005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.