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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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Dose-dependence of DiCre activation in vivo.A: Southern blots, using a LacZ probe, prepared from the liver of adult DiCreR26R animals ten days following 510mgkg, 310mgkg, 110mgkg, 520mgkg or 540mgkg i.p. rapamycin treatment, with two or three animals for each condition. The upper band R corresponds to the R26R allele after recombination, the lower band NR to the nonrecombined allele. B Quantification of the degree of recombination, as estimated by image analysis of the blot shown above.
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pone-0001355-g005: Dose-dependence of DiCre activation in vivo.A: Southern blots, using a LacZ probe, prepared from the liver of adult DiCreR26R animals ten days following 510mgkg, 310mgkg, 110mgkg, 520mgkg or 540mgkg i.p. rapamycin treatment, with two or three animals for each condition. The upper band R corresponds to the R26R allele after recombination, the lower band NR to the nonrecombined allele. B Quantification of the degree of recombination, as estimated by image analysis of the blot shown above.

Mentions: To evaluate the number of rapamycin injections needed to induce DiCre-mediated recombination, mice were treated with different drug regimens, i.e. daily injections of 10 mg/kg rapamycin for one, three or five days. In parallel, higher doses (5×20 or 5×40 mg/kg) of rapamycin were also tested. Animals were sacrificed ten days after the last injection and recombination rate was quantitatively evaluated in the liver using Southern blots. The results indicate that the degree of recombination is equivalent for five or three injections and somewhat lower with a single injection (Fig. 5). It should be noted also that the use of the highest dose of rapamycin (40 mg/kg) seems to increase the degree of recombination.


Conditional transgenesis using Dimerizable Cre (DiCre).

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

Dose-dependence of DiCre activation in vivo.A: Southern blots, using a LacZ probe, prepared from the liver of adult DiCreR26R animals ten days following 510mgkg, 310mgkg, 110mgkg, 520mgkg or 540mgkg i.p. rapamycin treatment, with two or three animals for each condition. The upper band R corresponds to the R26R allele after recombination, the lower band NR to the nonrecombined allele. B Quantification of the degree of recombination, as estimated by image analysis of the blot shown above.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001355-g005: Dose-dependence of DiCre activation in vivo.A: Southern blots, using a LacZ probe, prepared from the liver of adult DiCreR26R animals ten days following 510mgkg, 310mgkg, 110mgkg, 520mgkg or 540mgkg i.p. rapamycin treatment, with two or three animals for each condition. The upper band R corresponds to the R26R allele after recombination, the lower band NR to the nonrecombined allele. B Quantification of the degree of recombination, as estimated by image analysis of the blot shown above.
Mentions: To evaluate the number of rapamycin injections needed to induce DiCre-mediated recombination, mice were treated with different drug regimens, i.e. daily injections of 10 mg/kg rapamycin for one, three or five days. In parallel, higher doses (5×20 or 5×40 mg/kg) of rapamycin were also tested. Animals were sacrificed ten days after the last injection and recombination rate was quantitatively evaluated in the liver using Southern blots. The results indicate that the degree of recombination is equivalent for five or three injections and somewhat lower with a single injection (Fig. 5). It should be noted also that the use of the highest dose of rapamycin (40 mg/kg) seems to increase the degree of recombination.

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