Submitted on January 20, 2007
Accepted on April 4, 2007
Toxicity of ligand-dependant Cre-recombinases and generation of a conditional Cre-deleter mouse allowing mosaic recombination in peripheral tissues
Dorothe Hameyer1, Leonid Eshkind1, Steffen Schmitt2, Cecilia Antunes1, Annemiek Groen3, Eric Bindels4, Jos Jonkers5, Paul Krimpenfort4, Ralph Meuwissen4, Loes Rijswijk3, Axel Bex4, Anton Berns4, and Ernesto Bockamp1*
1 Institute of Toxicology/Mousegenetics, Johannes Gutenberg-Universität Mainz, Germany
2 FACS and Array Core Facility, Johannes Gutenberg-Universität Mainz, Mainz, Germany
3 Academic Medical Center, AMC Liver Center, The Netherlands Cancer Institute, Amsterdam, Netherlands; Division of Molecular Genetics and Center of Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, Netherlands
4 Division of Molecular Genetics and Center of Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, Netherlands
5 Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, Netherlands
* To whom correspondence should be addressed. E-mail: bockamp{at}mail.uni-mainz.de.
Ligand-activated Cre recombinases are widely used for studying gene function in vitro and in conditional mouse models. To compare ligand-dependent Cre recombinases, different Cre estrogen receptor fusions were introduced into the ROSA26 locus of embryonic stem (ES) cells and assayed for genotoxicity and recombination efficiency. Of the tested recombinases, the CreERT2 variant showed no toxicity and was highly responsive to ligand induction. To constitutively express CreERT2 in mice and to also clarify if the CreERT2 system displays background activity, we generated a knock-in mouse line harbouring the CreERT2 coding region under the control of the ROSA26 locus. Analysis of this ROSA26 CreERT2 deleter mouse with different reporter strains revealed ubiquitous recombination in the embryo, partial recombination in peripheral and haematopoietic tissues but no effective CreERT2 expression in the brain. Furthermore, using flow cytometry we found low level background recombination in non-induced bi-transgenic ROSA26 CreERT2/EGFP reporter mice. To determine whether background activity poses a general problem for conducting conditional in vivo experiments with the ROSA26 CreERT2 deleter, we used a sensitive conditional skin cancer model. In this assay cancer induction was completely restricted to induced bi-transgenic CreERT2/K-RasV12 mice whereas non-induced control animals did not show any sign of cancer indicating the usefulness of the ROSA CreERT2 system for regulating conditional gene expression in vivo. The ROSA26 CreERT2 deleter strain will be a convenient experimental tool for studying gene function under circumstances requiring partial induction of recombination in peripheral tissues and will be useful for uncovering previously unknown or unsuspected phenotypes.
