Conditional Transgenesis Using Dimerizable Cre (DiCre)

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 DiCre×R26R 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

The targeted inactivation of Men1 gene exacerbates Ret/PTC3-induced thyroid neoplastic transformation

Resumen del trabajo presentado al 38th Annual Meeting of the European Thyroid Association celebrado en Santiago de Compostela (España) del 6 al 10 de septiembre de 2014.-- et al.Multiple Endocrine Neoplasia type1 is an inherited predisposition syndrome characterized by an increased risk of endocrine tumors. In a previous study, we found that menin acts as a negative regulator controlling the thyroid growth. To explore the tumor suppressor function of menin, we analyzed whether thyroid-selective inactivation of Men1 gene affects Ret/PTC3-driven neoplastic transformation. Mice with Men1-disruption selectively in thyrocytes (Men1flox/floxThyrCre) were generated by crossing transgenic ThyrCre mice (expressing the Cre selectively in thyrocytes) with mice carrying Men1floxalleles. Men1flox/floxThyrCre mice were then crossed with mice expressing the Ret-PTC3 oncogene selectively in the thyroid (RP3mice). Thyroid phenotype of mice was analyzed at 4, 8 and 12 months of age. RP3-induced thyroid hypertrophy was transiently reduced and followed by a significant increase in aging Men1flox/floxThyrCre/RP3 mice in comparison with aged-matched RP3 littermates. At 4, 8 and12 months of age, hormonal status analyses disclosed a decrease of serum T4 and T3 associated with a marked increase in serum TSH concentrations. The functional activity of the thyroid assessed by measurements of the expression level of thyroid-specific genes appeared reduced in Men1flox/floxThyrCre/RP3 mice compared to that of age-matched RP3 littermates. In aging Men1flox/floxThyrCre/RP3 mice, morphological examinations of the thyroid showed a profound tissue remodeling associated with the occurrence of mesenchymal structures. This observation was evidenced by a 2-fold-increase in vimentin and fibronectin1 without significant decrease in E-cadherin mRNA levels. However, a 3-fold-increase in Sip1 and Snail mRNA levels known to be E-cadherin repressors involved in the epithelial-to-mesenchymal transition (EMT) was observed in Men1flox/ floxThyrCre/RP3 mice compared to that of RP3 littermates. Our data indicate that tumors developped following Ret/PTC3 expression are susceptible to undergo EMT in response to the targeted-inactivation of the Men1 gene. In conclusion, we show for the first time that Men1 gene acts as a tumor suppressor in Ret/PTC3-driven thyroid neoplastic transformation.Peer Reviewe

Regulation of Cre recombinase by ligand-induced complementation of inactive fragments

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. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12–rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05–0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48–72 h, with an EC(50) of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals

The Targeted Inactivation of TRβ Gene in Thyroid Follicular Cells Suggests a New Mechanism of Regulation of Thyroid Hormone Production

International audienceThyroid epithelial cells, or thyrocytes, express functional thyroid hormone receptors but no precise role has yet been assigned to either TRα or TRβ in the thyroid gland. In this study, we analyzed the impact of inactivating the TRβ gene in the thyroid of mice. First, we generated a mouse line named Thyr-Cre, expressing the Cre recombinase under the control of the thyroglobulin gene promoter, which led to a complete recombination of floxed genes in thyrocytes. Thyr-Cre mice were then crossed with TRβ floxed mice (TRβflox/flox) to obtain a thyrocyte-selective deletion of TRβ. Thyr-TRβ−/− mice were characterized by a decrease in the size and functional activity of the thyroid gland. These alterations were associated with a decrease in plasma TSH concentration. Surprisingly, Thyr-TRβ−/− displayed elevated serum T4 and rT3 concentrations with no significant change in serum T3 levels. Their intrathyroidal free T4 and rT3 contents were also elevated, whereas the ratio of serum T4 to thyroid free T4 was decreased by comparison with wild-type littermates. Also, within the thyroid, deiodinases D1 and D2 were reduced as well as the expression levels of genes encoding monocarboxylate transporters (Mct8 and Mct10). Such a decrease in intrathyroidal deiodination of T4 and in the expression of genes encoding thyroid hormone transporters may contribute to the primary overproduction of T4 observed in Thyr-TRβ−/− mice. In conclusion, these data show that the control of thyroid hormone production involves not only TRβ-dependent mechanisms acting at the level of hypothalamus and pituitary but also TRβ-dependent mechanisms acting at the thyroid level

Histopathological changes induced by selective inactivation of menin on the thyroid gland in RET/PTC3 and E7 transgenic mice. A study of 77 cases

International audienceMultiple Endocrine Neoplasia Type 1 (MEN1) does not involve the thyroid gland, but animal studies have shown that mice with inactivation of menin could develop thyroid pathologies. The objective was to evaluate if the selective inactivation of menin in murine thyroid glands expressing RET/PTC3 and E7 oncogenes, might induce an increased index of proliferation and a more rapid development of thyroid hyperplasia and/or tumors. The thyroid glands of 77 mice aged 4-18 months (31 expressing the E7 oncogene and 25 the RET/PTC3 oncogene) were analyzed for histological changes and Ki67 proliferation index. Fifty-two mice had selective inactivation of menin in the thyroid gland (16 mice with RET/PTC3 oncogene and 19 mice with E7 oncogene). As compared to wild type, mice with inactivation of menin presented an increased Ki67 proliferation index. Mice presenting the E7 oncogene showed larger thyroid glands with a pattern of diffuse hyperplasia. Mice expressing the RET/PTC3 oncogene presented larger thyroid glands compared to the wild type mice but smaller compared to E7 mice. The lesions in the RET/PTC3 group were "proliferative papillary cystic changes" (60%), "cribriform" (16%), "solid" (8%) and a combination of these patterns in the rest of the thyroid glands. The inactivation of menin in the thyroid gland of young mice does not seem to change the histological pattern, but it influences the proliferation of follicular cells. Further molecular studies especially in aged mice are needed to better understand the correlation between certain oncogenes and the inactive status of meni

Expression of the DiCre constructs in tissues of adult or embryonic transgenic animals.

<p>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.</p

Induction of Cre-mediated recombination in DiCre×Z/EG mice.

<p>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.</p

Induction of Cre-mediated recombination in various tissues of DiCre×R26R mice.

<p>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.</p