Characterization of parameters required for effective use of tamoxifen-regulated recombination.
Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems. Ligand regulated forms of Cre have been increasingly used in these applications in order to gain temporal and spatial control over conditional targeting. The tamoxifen-regulated Cre variant mer-Cre-mer (mCrem) is widely utilized because of its reputation for tight regulation in the absence of its tamoxifen ligand. In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem. This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target. We find that robust mCrem expression correlates with a high level of tamoxifen-independent Cre activity, while clones expressing mCrem at the limit of western blot detection exhibit extremely tight regulation. We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population.
Buelow
Ben
B
Department of Pediatrics, University of Washington, Seattle Children's Hospital Research Institute, Seattle, Washington, USA.
Scharenberg
Andrew M
AM
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D001483:Base Sequence; D002645:Chickens; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D018390:Gene Targeting; D019426:Integrases / Q000378:metabolism; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology*; D019076:Transgenes
D008024:Ligands; D020845:Selective Estrogen Receptor Modulators; D013629:Tamoxifen; C045073:Cre recombinase; D019426:Integrases