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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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Related in: MedlinePlus

Targeting strategy for the creation of DiCre ES cells.A: Scheme of the targeting vector and of the Rosa26 locus following homologous recombination. “B” and “R” stand, respectively, for the BamHI and EcoRV sites that are used when screening ES cells clones for correct insertions using Southern blots and the probes indicated on the scheme. Fragments amplified when genotyping animals are also represented B: Representative examples of Southern blots obtained from wild type ES cells or clones with correct recombination, using the probes shown on the schemes above.
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pone-0001355-g001: Targeting strategy for the creation of DiCre ES cells.A: Scheme of the targeting vector and of the Rosa26 locus following homologous recombination. “B” and “R” stand, respectively, for the BamHI and EcoRV sites that are used when screening ES cells clones for correct insertions using Southern blots and the probes indicated on the scheme. Fragments amplified when genotyping animals are also represented B: Representative examples of Southern blots obtained from wild type ES cells or clones with correct recombination, using the probes shown on the schemes above.

Mentions: Details of the targeting construct are given on Figure 1A. Following its successful insertion, the C-terminal component of DiCre is expressed under the control of the Rosa-26 promoter, while the expression of the N-terminal component is controlled by the strong CAG promoter. In light of the ubiquitous expression and the absence of silencing of genes expressed from this locus, documented both during development and following birth [30], it was felt that this approach would ensure a generalized and reliable expression of DiCre. Note that a second promoter (PGK) with a downstream hygromycin-resistance gene, used for selection of successfully targeted ES cells, will also be present in the locus following targeting. However, this cassette has been removed secondarily from the genome by crossing F1 animals with Flpe-expressing mice [31] and is not present in the final lines presented here.


Conditional transgenesis using Dimerizable Cre (DiCre).

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

Targeting strategy for the creation of DiCre ES cells.A: Scheme of the targeting vector and of the Rosa26 locus following homologous recombination. “B” and “R” stand, respectively, for the BamHI and EcoRV sites that are used when screening ES cells clones for correct insertions using Southern blots and the probes indicated on the scheme. Fragments amplified when genotyping animals are also represented B: Representative examples of Southern blots obtained from wild type ES cells or clones with correct recombination, using the probes shown on the schemes above.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001355-g001: Targeting strategy for the creation of DiCre ES cells.A: Scheme of the targeting vector and of the Rosa26 locus following homologous recombination. “B” and “R” stand, respectively, for the BamHI and EcoRV sites that are used when screening ES cells clones for correct insertions using Southern blots and the probes indicated on the scheme. Fragments amplified when genotyping animals are also represented B: Representative examples of Southern blots obtained from wild type ES cells or clones with correct recombination, using the probes shown on the schemes above.
Mentions: Details of the targeting construct are given on Figure 1A. Following its successful insertion, the C-terminal component of DiCre is expressed under the control of the Rosa-26 promoter, while the expression of the N-terminal component is controlled by the strong CAG promoter. In light of the ubiquitous expression and the absence of silencing of genes expressed from this locus, documented both during development and following birth [30], it was felt that this approach would ensure a generalized and reliable expression of DiCre. Note that a second promoter (PGK) with a downstream hygromycin-resistance gene, used for selection of successfully targeted ES cells, will also be present in the locus following targeting. However, this cassette has been removed secondarily from the genome by crossing F1 animals with Flpe-expressing mice [31] and is not present in the final lines presented here.

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