Conditional Transgenesis Using Dimerizable Cre (DiCre)

Abstract

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCre×R26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters.

Spatiotemporal Control of Vascular Endothelial Growth Factor Expression Using a Heat-Shock-Activated, Rapamycin-Dependent Gene Switch

“…Detailed in vitro characterization of C3HT101/2-fLuc and VEGF cells showed strong thermal inducibility of the gene switch in the presence of nanomolar concentrations of heterodimerizer, but negligible transgene expression when one or both stimuli are absent. Consistent with previous reports ( Jullien et al, 2007), we found the activity of AP21967 to be approximately one-fifth the activity of rapamycin. However, this compound does not inhibit endogenous mTOR and has less than one thousandth the immunosuppressive activity of rapamycin (Indraccolo et al, 2006).…”

Novel Thioredoxin-Like Proteins Are Components of a Protein Complex Coating the Cortical Microtubules of Toxoplasma gondii

“…If the target gene is essential and the knockout mutant fails to grow as a clone, one can still use the mixed population of the Cre-transfected LoxP knock-in line to identify the mutants under the microscope based on fluorescence and analyze the knockout phenotype in live cells. This strategy can be used in any Cre-LoxP-based technique regardless of how Cre expression is controlled (i.e., by transient transfection as described previously [29,30] and in this work or by conditional expression, as in the case of the DiCre system first developed for mouse genetics [46] and recently implemented in T. gondii and P. falciparum [47,48]). …”

Cre Recombinase

“…The small molecule rapamycin induces heterodimerization of FKBP and FRB (110), leading to reconstitution of an active Cre subunit. This method of regulation has been demonstrated to function in vitro (109) as well as in mice (111).…”