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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 various tissues of DiCre×R26R mice.The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in various tissues of adult DiCre×R26R animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). Bars represent 100 µm. The insert for the liver shows the total absence of recombination in the absence of rapamycin treatment.
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pone-0001355-g004: Induction of Cre-mediated recombination in various tissues of DiCre×R26R mice.The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in various tissues of adult DiCre×R26R animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). Bars represent 100 µm. The insert for the liver shows the total absence of recombination in the absence of rapamycin treatment.

Mentions: To control and complement these results using another indicator mouse line, DiCre mice were mated with R26R animals [29] and the existence of recombination examined on tissue sections by X-gal staining. Again, no labeled cells were observed in the absence of treatment. On the other hand, and contrary to what had been observed with the Z/EG indicator line, recombination was widespread and could be observed in a number of tissues after treatment of the in DiCre×R26R animals with the inducer (Fig. 4). Thus, blue cells could be observed in heart, kidney, liver, testis, adrenals, fat tissue (not shown), lung, pituitary (adenohypohysis), spleen. However, the degree of staining differed greatly among these tissues, being quite dense in the liver where up to 30–40% of the cells were blue, intermediate in the heart and kidney, and low (only a few cells per sections) in the other tissues. Moreover, staining was not necessarily uniform in tissues with complex architecture, such as in the kidney where staining was more concentrated in the region of renal papilla and much less in the cortex, in the lung with staining detectable only in the alveolar epithelium, or in the testis, where only interstitial Leydig cells showed staining. No staining could be observed in the muscle, brain, or epidermis. The pattern of recombination was the same when higher doses of rapamycin were used (up to 40 mg/kg, results not shown).


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 various tissues of DiCre×R26R mice.The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in various tissues of adult DiCre×R26R animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). Bars represent 100 µm. The insert for the liver shows the total absence of recombination in the absence of rapamycin treatment.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001355-g004: Induction of Cre-mediated recombination in various tissues of DiCre×R26R mice.The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in various tissues of adult DiCre×R26R animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). Bars represent 100 µm. The insert for the liver shows the total absence of recombination in the absence of rapamycin treatment.
Mentions: To control and complement these results using another indicator mouse line, DiCre mice were mated with R26R animals [29] and the existence of recombination examined on tissue sections by X-gal staining. Again, no labeled cells were observed in the absence of treatment. On the other hand, and contrary to what had been observed with the Z/EG indicator line, recombination was widespread and could be observed in a number of tissues after treatment of the in DiCre×R26R animals with the inducer (Fig. 4). Thus, blue cells could be observed in heart, kidney, liver, testis, adrenals, fat tissue (not shown), lung, pituitary (adenohypohysis), spleen. However, the degree of staining differed greatly among these tissues, being quite dense in the liver where up to 30–40% of the cells were blue, intermediate in the heart and kidney, and low (only a few cells per sections) in the other tissues. Moreover, staining was not necessarily uniform in tissues with complex architecture, such as in the kidney where staining was more concentrated in the region of renal papilla and much less in the cortex, in the lung with staining detectable only in the alveolar epithelium, or in the testis, where only interstitial Leydig cells showed staining. No staining could be observed in the muscle, brain, or epidermis. The pattern of recombination was the same when higher doses of rapamycin were used (up to 40 mg/kg, results not shown).

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