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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 DiCrexR26R 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.

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. 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 efficiently heterodimerized 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 EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently 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 knock-out animals.

To prepare immortalized adrenal chromaffin cells for eventual clinical use, the immortalizing oncogene must be removed. We have utilized a Cre-mediated excision of a loxP-flanked Tag sequence to test whether immortalized chromaffin cells could be disimmortalized by this method. Cultures of embryonic rat adrenal cells were immortalized with the tsA-TN retroviral vector encoding the loxP-flanked temperature-sensitive allele of SV40 large T antigen (tsA-TN) and a positive/negative neo/HSV-TK sequence for selection with either G418 or gancyclovir, respectively. These cells were then infected with the 1710-CrePR1 bicistronic retroviral vector coding for a form of Cre modulatable by the synthetic steroid RU486. These immortalized loxTsTag/CrePR1/RAD cells expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). After initial incubation at 37 degrees C with RU486 for 3 days, followed by the addition of gancyclovir for 7 days, Tag-ir was not detectable in most of the surviving chromaffin cells, compared to 100% expression in immortalized loxTsTag/CreR1/RAD cells not treated with RU486 and gancyclovir. The expression of TH, DbetaH, and PNMT was increased after disimmortalization and the ability of disimmortalized cells to synthesize norepinephrine was also significantly increased compared to immortalized cells. When both types of chromaffin cells were transplanted in a model of neuropathic pain and partial nerve injury, both cell grafts were equally able to reverse the behavioral hypersensitivity induced by the injury. The use of Cre/lox site-directed disimmortalization of chromaffin cells that are able to deliver neuroactive molecules offers a novel approach to cell therapy., (Copyright 2002 Elsevier Science (USA).)