A highly efficient ligand‐regulated Cre recombinase mouse line shows that LoxP recombination is position dependent

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone‐binding domain (ERT) to keep Cre inactive unless the non‐steroidal estrogen analog 4‐hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre‐ERT from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.

[1]  J. Vonesch,et al.  Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  A. Berns,et al.  Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. , 2000, Genes & development.

[3]  N. Henderson,et al.  Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[4]  E. Fuchs,et al.  The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P Chambon,et al.  Ligand-activated site-specific recombination in mice. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[6]  T. Noda,et al.  Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene. , 1997, Science.

[7]  L. Bennett,et al.  Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice , 1998, Oncogene.

[8]  S. Dymecki.  Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[9]  G. Wahl,et al.  Recombinase-mediated gene activation and site-specific integration in mammalian cells. , 1991, Science.

[10]  David Garrick,et al.  Repeat-induced gene silencing in mammals , 1998, Nature Genetics.

[11]  A. Berns,et al.  Cre-mediated somatic site-specific recombination in mice. , 1997, Nucleic acids research.

[12]  R Kuhn,et al.  Temporally and spatially regulated somatic mutagenesis in mice. , 1998, Nucleic acids research.

[13]  K. Rajewsky,et al.  Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting , 1993, Cell.

[14]  A. McMahon,et al.  Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase , 1998, Current Biology.

[15]  H. Niwa,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector. , 1991, Gene.

[16]  A. Berns,et al.  Requirement for a functional Rb-1 gene in murine development , 1992, Nature.

[17]  J. Lindeberg,et al.  Conditional gene targeting. , 2003, Upsala journal of medical sciences.

[18]  A. Bradley,et al.  Murine Brca2: sequence, map position, and expression pattern. , 1997, Genomics.

[19]  S. Robinson,et al.  Metabolites, pharmacodynamics, and pharmacokinetics of tamoxifen in rats and mice compared to the breast cancer patient. , 1991, Drug metabolism and disposition: the biological fate of chemicals.

[20]  S. Henikoff.  Conspiracy of silence among repeated transgenes. , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[21]  M. Vooijs,et al.  Developmental defects and tumor predisposition in Rb mutant mice , 1999, Oncogene.

[22]  Philippe Soriano.  Generalized lacZ expression with the ROSA26 Cre reporter strain , 1999, Nature Genetics.

[23]  M. Rudnicki,et al.  Simplified mammalian DNA isolation procedure. , 1991, Nucleic acids research.

[24]  R. Hammer,et al.  Sustained somatic gene inactivation by viral transfer of Cre recombinase , 1996, Nature Biotechnology.

[25]  L A Herzenberg,et al.  Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[27]  M Aguet,et al.  Inducible gene targeting in mice , 1995, Science.

[28]  A. Porter.  Controlling your losses: conditional gene silencing in mammals. , 1998, Trends in Genetics.

[29]  K. Rajewsky,et al.  Conditional gene targeting. , 1996, The Journal of clinical investigation.

[30]  L. Tsai,et al.  Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Karen Holbrook,et al.  Calcium regulation of growth and differentiation of mouse epidermal cells in culture , 1980, Cell.

[32]  M. Vooijs,et al.  Flp-mediated tissue-specific inactivation of the retinoblastoma tumor suppressor gene in the mouse , 1998, Oncogene.

[33]  D. Metzger,et al.  Engineering the mouse genome by site-specific recombination. , 1999, Current opinion in biotechnology.

[34]  Wen-Hwa Lee,et al.  Temporal, spatial, and cell type–specific control of Cre-mediated DNA recombination in transgenic mice , 1999, Nature Biotechnology.

[35]  G M Edelman,et al.  Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene. , 1996, Proceedings of the National Academy of Sciences of the United States of America.