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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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Expression of the DiCre constructs in tissues of adult or embryonic transgenic animals.A: Northern blots obtained from DiCre ES cells or tissues from young adult DiCre transgenic animals (two independent samples for each), indicating the level of mRNA for the indicated transgenes. B: Expression level, relative to that measured in the liver, of the same transgenes in tissues of adult animals, as measured by qPCR (means +/− SEM, n = 3–5). C: Northern blot, and D: qPCR analysis, as in panels 7A and 7B, from embryonic (E15) tissues and adult tissues used as comparison. Note that the adult tissues used here were not the same as those shown on panels 7A and 7B.
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pone-0001355-g007: Expression of the DiCre constructs in tissues of adult or embryonic transgenic animals.A: Northern blots obtained from DiCre ES cells or tissues from young adult DiCre transgenic animals (two independent samples for each), indicating the level of mRNA for the indicated transgenes. B: Expression level, relative to that measured in the liver, of the same transgenes in tissues of adult animals, as measured by qPCR (means +/− SEM, n = 3–5). C: Northern blot, and D: qPCR analysis, as in panels 7A and 7B, from embryonic (E15) tissues and adult tissues used as comparison. Note that the adult tissues used here were not the same as those shown on panels 7A and 7B.

Mentions: Evaluation of the level of transcripts, using Northern blots (Fig. 7A) or qPCR (Fig. 7B), in ES cells clones used to establish the DiCre mouse lines or in different tissues from young adult DiCre mice indicated that the level of transcription of Cre60.F2 from the Rosa26 promoter was relatively homogeneous and at comparable levels among these different tissues, except for the brain in which transcription seemed to be somewhat lower. For Cre59.F2 the level of expression, driven by the CAG promoter, was considerably higher than that mediated by the Rosa26 promoter for Cre60.F2. However, while for the Cre59.F2 transcript levels in the liver and ES cells were similar, the levels observed in the skin, kidney and brain were considerably lower. In tissues from E15 embryos, the level of Cre60.F2 transcription was diminished relative to the adult levels and transcription of Cre59.F2, controlled by the CAG promoter, was also found to be considerably decreased (Fig. 7C and 7D).


Conditional transgenesis using Dimerizable Cre (DiCre).

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

Expression of the DiCre constructs in tissues of adult or embryonic transgenic animals.A: Northern blots obtained from DiCre ES cells or tissues from young adult DiCre transgenic animals (two independent samples for each), indicating the level of mRNA for the indicated transgenes. B: Expression level, relative to that measured in the liver, of the same transgenes in tissues of adult animals, as measured by qPCR (means +/− SEM, n = 3–5). C: Northern blot, and D: qPCR analysis, as in panels 7A and 7B, from embryonic (E15) tissues and adult tissues used as comparison. Note that the adult tissues used here were not the same as those shown on panels 7A and 7B.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001355-g007: Expression of the DiCre constructs in tissues of adult or embryonic transgenic animals.A: Northern blots obtained from DiCre ES cells or tissues from young adult DiCre transgenic animals (two independent samples for each), indicating the level of mRNA for the indicated transgenes. B: Expression level, relative to that measured in the liver, of the same transgenes in tissues of adult animals, as measured by qPCR (means +/− SEM, n = 3–5). C: Northern blot, and D: qPCR analysis, as in panels 7A and 7B, from embryonic (E15) tissues and adult tissues used as comparison. Note that the adult tissues used here were not the same as those shown on panels 7A and 7B.
Mentions: Evaluation of the level of transcripts, using Northern blots (Fig. 7A) or qPCR (Fig. 7B), in ES cells clones used to establish the DiCre mouse lines or in different tissues from young adult DiCre mice indicated that the level of transcription of Cre60.F2 from the Rosa26 promoter was relatively homogeneous and at comparable levels among these different tissues, except for the brain in which transcription seemed to be somewhat lower. For Cre59.F2 the level of expression, driven by the CAG promoter, was considerably higher than that mediated by the Rosa26 promoter for Cre60.F2. However, while for the Cre59.F2 transcript levels in the liver and ES cells were similar, the levels observed in the skin, kidney and brain were considerably lower. In tissues from E15 embryos, the level of Cre60.F2 transcription was diminished relative to the adult levels and transcription of Cre59.F2, controlled by the CAG promoter, was also found to be considerably decreased (Fig. 7C and 7D).

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