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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 DiCre×Z/EG mice.A: The presence of recombination is shown by EGFP expression in the heart of DiCre×Z/EG adult animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). B: In the absence of rapamycin treatment no specific fluorescence can be seen.
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pone-0001355-g003: Induction of Cre-mediated recombination in DiCre×Z/EG mice.A: The presence of recombination is shown by EGFP expression in the heart of DiCre×Z/EG adult animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). B: In the absence of rapamycin treatment no specific fluorescence can be seen.

Mentions: In a first series of test, DiCre mice were mated with Z/EG transgenic indicator mice to obtain double transgenic mice. The Z/EG transgene is similar to the CALNLZ construct depicted on Fig. 2A, except for the stuffer sequence that codes for LacZ and the gene expressed following the excision of the stuffer sequence that is EGFP [33]. Thus, cells of these animals are LacZ+ in the absence of recombination and green fluorescent following Cre-mediated excision. Young adult double transgenic progenies resulting from this mating were treated with the inducer (daily injection of 10 mg/kg rapamycin for five days) and sacrificed 5 days after the end of the treatment. In the absence of treatment no fluorescent cells could be detected on tissue sections prepared from a number of tissues (brain, pituitary, heart, lung, liver, kidney, skin, striated muscle). In treated animals EGFP-positive (as confirmed by immunohistochemistry, results not shown) green fluorescent fibers, indicating that recombination has taken place, were present only in the heart, and surprisingly, recombined green fluorescent cells could be detected in no other tissues (Fig. 3). Because of the low number of recombined cells, this switch on of EGFP expression could not be correlated to a significant decrease of the level of X-gal staining in the heart. Note that in untreated animals, strong and definite X-gal staining was observable only in the heart and muscle.


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 DiCre×Z/EG mice.A: The presence of recombination is shown by EGFP expression in the heart of DiCre×Z/EG adult animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). B: In the absence of rapamycin treatment no specific fluorescence can be seen.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001355-g003: Induction of Cre-mediated recombination in DiCre×Z/EG mice.A: The presence of recombination is shown by EGFP expression in the heart of DiCre×Z/EG adult animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). B: In the absence of rapamycin treatment no specific fluorescence can be seen.
Mentions: In a first series of test, DiCre mice were mated with Z/EG transgenic indicator mice to obtain double transgenic mice. The Z/EG transgene is similar to the CALNLZ construct depicted on Fig. 2A, except for the stuffer sequence that codes for LacZ and the gene expressed following the excision of the stuffer sequence that is EGFP [33]. Thus, cells of these animals are LacZ+ in the absence of recombination and green fluorescent following Cre-mediated excision. Young adult double transgenic progenies resulting from this mating were treated with the inducer (daily injection of 10 mg/kg rapamycin for five days) and sacrificed 5 days after the end of the treatment. In the absence of treatment no fluorescent cells could be detected on tissue sections prepared from a number of tissues (brain, pituitary, heart, lung, liver, kidney, skin, striated muscle). In treated animals EGFP-positive (as confirmed by immunohistochemistry, results not shown) green fluorescent fibers, indicating that recombination has taken place, were present only in the heart, and surprisingly, recombined green fluorescent cells could be detected in no other tissues (Fig. 3). Because of the low number of recombined cells, this switch on of EGFP expression could not be correlated to a significant decrease of the level of X-gal staining in the heart. Note that in untreated animals, strong and definite X-gal staining was observable only in the heart and muscle.

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