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Conditional transgenesis using Dimerizable Cre (DiCre).

Jullien N, Goddard I, Selmi-Ruby S, Fina JL, Cremer H, Herman JP - PLoS ONE (2007)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: ICNE-UMR 6544 Centre National de Recherche Scientifique (CNRS), Université de Méditerranée, Marseille, France.

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 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.

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Induction of Cre-mediated recombination in primary cultures of embryonic tissues of DiCre×R26R mice.The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product on the bright-field (upper row) and phase contrast (lower row) images of the same fields, in primary cultures prepared from various tissues of DiCre×R26R E15 embryos. Cultures were exposed between 3 and 7 day in vitro to 10 nM AP23102.
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pone-0001355-g006: Induction of Cre-mediated recombination in primary cultures of embryonic tissues of DiCre×R26R mice.The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product on the bright-field (upper row) and phase contrast (lower row) images of the same fields, in primary cultures prepared from various tissues of DiCre×R26R E15 embryos. Cultures were exposed between 3 and 7 day in vitro to 10 nM AP23102.

Mentions: To test whether the absence of recombination in embryos could be due to an insufficient passage of the dimerizer through the placental barrier, primary cultures were prepared from CNS, liver, heart of E15 DiCre×R26R embryos. Cultures were also prepared from the carcass of embryos using protocols described for obtention of Mouse Embryonic Fibroblasts [34] resulting in a mixed primary culture we call “MEF” and that contains, besides fibroblasts, other types of cells, such as myocytes and fused muscle fibers etc. Cre activity was induced by adding 10 nM AP23102 (DiCre×R26R cells) at 3 DIV and LacZ activity was tested four days later. Results were similar to what had been observed in vivo, i.e. some recombined cells could be observed among hepatocytes or cardiomyocytes obtained from DiCre×R26R embryos, but their proportion was very low (Fig. 6.).


Conditional transgenesis using Dimerizable Cre (DiCre).

Jullien N, Goddard I, Selmi-Ruby S, Fina JL, Cremer H, Herman JP - PLoS ONE (2007)

Induction of Cre-mediated recombination in primary cultures of embryonic tissues of DiCre×R26R mice.The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product on the bright-field (upper row) and phase contrast (lower row) images of the same fields, in primary cultures prepared from various tissues of DiCre×R26R E15 embryos. Cultures were exposed between 3 and 7 day in vitro to 10 nM AP23102.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2131782&req=5

pone-0001355-g006: Induction of Cre-mediated recombination in primary cultures of embryonic tissues of DiCre×R26R mice.The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product on the bright-field (upper row) and phase contrast (lower row) images of the same fields, in primary cultures prepared from various tissues of DiCre×R26R E15 embryos. Cultures were exposed between 3 and 7 day in vitro to 10 nM AP23102.
Mentions: To test whether the absence of recombination in embryos could be due to an insufficient passage of the dimerizer through the placental barrier, primary cultures were prepared from CNS, liver, heart of E15 DiCre×R26R embryos. Cultures were also prepared from the carcass of embryos using protocols described for obtention of Mouse Embryonic Fibroblasts [34] resulting in a mixed primary culture we call “MEF” and that contains, besides fibroblasts, other types of cells, such as myocytes and fused muscle fibers etc. Cre activity was induced by adding 10 nM AP23102 (DiCre×R26R cells) at 3 DIV and LacZ activity was tested four days later. Results were similar to what had been observed in vivo, i.e. some recombined cells could be observed among hepatocytes or cardiomyocytes obtained from DiCre×R26R embryos, but their proportion was very low (Fig. 6.).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: ICNE-UMR 6544 Centre National de Recherche Scientifique (CNRS), Université de Méditerranée, Marseille, France.

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 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.

Show MeSH
Related in: MedlinePlus