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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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DiCre are functional in targeted ES cells.A: Scheme of the CALNLZ indicator construct used to test the functionality of Cre. B: ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in ES cells with correct insertion of the DiCre construct into the Rosa26 locus and stable transfection of the pcDNA-CALNLZ construct. Cells are tested without (“Control”) or following exposure, for three days, to 20 nM of the dimerizer AP23102.
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pone-0001355-g002: DiCre are functional in targeted ES cells.A: Scheme of the CALNLZ indicator construct used to test the functionality of Cre. B: ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in ES cells with correct insertion of the DiCre construct into the Rosa26 locus and stable transfection of the pcDNA-CALNLZ construct. Cells are tested without (“Control”) or following exposure, for three days, to 20 nM of the dimerizer AP23102.

Mentions: Following the obtention of clones of successfully targeted ES cells and before using them for blastocyst injection to obtain conditional deleter mice, we wished to test that DiCre was functional in these clones. Cells were transfected with an indicator construct, pcDNA3-CALNLZ [32] that expresses the ß-galactosidase reporter gene in transfected cells conditionally, dependent on the Cre-mediated excision of a neomycin resistance-STOP-pA cassette inserted between it and a CAG promoter (Fig. 2A). Following G418 selection, we obtained several subclones for the DiCre-containing ES clones. As shown on Figure 2B, there was a complete lack of expression of ß-galactosidase in the cells in the absence of inducer, indicating that background activity in these conditions is even lower than that we have observed earlier in fibroblasts. On the other hand, exposure of the cells to the inducer (20 nM of the rapamycin analog AP23102 for three days) led to the appearance of ß-galactosidase activity in close to 100% of undifferentiated cells, indicating that the system does work in ES cells. Note, however, that ß-galactosidase activity was lower or absent in flat cells, presumably engaged in some kind of differentiation pathway and surrounding the compact aggregates of undifferentiated ES cell. This aspect has not been pursued further.


Conditional transgenesis using Dimerizable Cre (DiCre).

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

DiCre are functional in targeted ES cells.A: Scheme of the CALNLZ indicator construct used to test the functionality of Cre. B: ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in ES cells with correct insertion of the DiCre construct into the Rosa26 locus and stable transfection of the pcDNA-CALNLZ construct. Cells are tested without (“Control”) or following exposure, for three days, to 20 nM of the dimerizer AP23102.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001355-g002: DiCre are functional in targeted ES cells.A: Scheme of the CALNLZ indicator construct used to test the functionality of Cre. B: ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in ES cells with correct insertion of the DiCre construct into the Rosa26 locus and stable transfection of the pcDNA-CALNLZ construct. Cells are tested without (“Control”) or following exposure, for three days, to 20 nM of the dimerizer AP23102.
Mentions: Following the obtention of clones of successfully targeted ES cells and before using them for blastocyst injection to obtain conditional deleter mice, we wished to test that DiCre was functional in these clones. Cells were transfected with an indicator construct, pcDNA3-CALNLZ [32] that expresses the ß-galactosidase reporter gene in transfected cells conditionally, dependent on the Cre-mediated excision of a neomycin resistance-STOP-pA cassette inserted between it and a CAG promoter (Fig. 2A). Following G418 selection, we obtained several subclones for the DiCre-containing ES clones. As shown on Figure 2B, there was a complete lack of expression of ß-galactosidase in the cells in the absence of inducer, indicating that background activity in these conditions is even lower than that we have observed earlier in fibroblasts. On the other hand, exposure of the cells to the inducer (20 nM of the rapamycin analog AP23102 for three days) led to the appearance of ß-galactosidase activity in close to 100% of undifferentiated cells, indicating that the system does work in ES cells. Note, however, that ß-galactosidase activity was lower or absent in flat cells, presumably engaged in some kind of differentiation pathway and surrounding the compact aggregates of undifferentiated ES cell. This aspect has not been pursued further.

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