Split-CreERT2: temporal control of DNA recombination mediated by split-Cre protein fragment complementation.
BACKGROUND
DNA recombination technologies such as the Cre/LoxP system advance modern biological research by allowing conditional gene regulation in vivo. However, the precise targeting of a particular cell type at a given time point has remained challenging since spatial specificity has so far depended exclusively on the promoter driving Cre recombinase expression. We have recently established split-Cre that allows DNA recombination to be controlled by coincidental activity of two promoters, thereby increasing spatial specificity of Cre-mediated DNA recombination. To allow temporal control of split-Cre-mediated DNA recombination we have now extended split-Cre by fusing split-Cre proteins with the tamoxifen inducible ERT2 domain derived from CreERT2.


RESULTS
In the split-CreERT2 system, Cre-mediated DNA recombination is controlled by two expression cassettes as well as the time of tamoxifen application. By using two independent Cre-dependent reporters in cultured cells, the combination of NCre-ERT2+ERT2-CCre was identified as having the most favorable properties of all constructs tested, showing an induction ratio of about 10 and EC(50)-values for 4-hydroxy-tamoxifen of 10 nM to 70 nM.


CONCLUSIONS
These characteristics of split-CreERT2 in vitro indicate that split-CreERT2 will be well suited for inducing DNA recombination in living mice harboring LoxP-flanked alleles. In this way, split-CreERT2 will provide a new tool of modern genetics allowing spatial and temporal precise genetic access to cell populations defined by the simultaneous activity of two promoters.
Hirrlinger
Johannes
J
N05 Neural Plasticity, Interdisciplinary Centre for Clinical Research (IZKF), University of Leipzig, Leipzig, Germany. johannes.hirrlinger@medizin.uni-leipzig.de
Requardt
Robert P
RP
Winkler
Ulrike
U
Wilhelm
Franziska
F
Schulze
Christine
C
Hirrlinger
Petra G
PG
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics*; D057075:Enzyme Assays; D005434:Flow Cytometry; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D019426:Integrases / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D051381:Rats; D011959:Receptors, Estradiol / Q000737:chemistry* / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D013997:Time Factors
D011959:Receptors, Estradiol; D004247:DNA; D008156:Luciferases; C045073:Cre recombinase; D019426:Integrases