Regulation of Cre recombinase by ligand-induced complementation of inactive fragments
authors
abstract
Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an ef®cient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12±rapamycin-associated protein), respectively. These can be ef®ciently hetero-dimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05±0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48±72 h, with an EC 50 of 0.02 nM. Thus, ligand-induced dimerization can ef®ciently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knockout animals.
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