Structures et sequences des genes de la caseine beta et de la WAP de lapin. Recherche 'in vitro' et 'in vivo' d'elements regulateurs sur le gene de la WAP

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : TD 80970 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Transgenesis for the study and the control of lactation

The study and the control of milk synthesis are required to decipher the mechanisms of gene expression, to improve milk production, to modify milk composition, to induce a resistance to diseases in the mammary gland and to produce recombinant proteins of pharmaceutical interest. Transgenesis has become a mandatory tool to reach these goals. The use of transgenesis is still limited by the difficulty of adding foreign genes in farm animals and mainly by replacing genes by homologous recombination. Transgene expression is also often ill-controlled. The present paper summarizes the current progress in this field with a particular emphasis on expression vectors for transgenes.La transgénèse pour l'étude et le contrôle de la lactation. L'étude et le contrôle de la synthèse du lait sont nécessaires pour décrypter les mécanismes de l'expression de gènes, pour améliorer la production de lait, pour modifier la composition du lait, pour induire une résistance de la glande mammaire contre des maladies infectieuses et pour produire des protéines recombinantes d'intérêt pharmaceutique. La transgénèse est devenue un outil indispensable pour mener à bien ces projets. La mise en œuvre de la transgénèse est encore limitée par la difficulté d'ajouter des gènes étrangers chez les animaux d'élevage et surtout de remplacer des gènes par recombinaison homologue. L'expression des transgènes est par ailleurs souvent mal contrôlée. Cet article se propose de faire le point dans ce domaine en développant plus particulièrement la mise au point de vecteurs d'expression pour les transgènes

The paralogs’ enigma of germ-cell specific genes dispensable for fertility: the case of 19 oogenesin genes

International audienceAbstract Gene knockout experiments have shown that many genes are dispensable for a given biological function. In this review, we make an assessment of male and female germ cell-specific genes dispensable for the function of reproduction in mice, the inactivation of which does not affect fertility. In particular, we describe the deletion of a 1 Mb block containing nineteen paralogous genes of the oogenesin/Pramel family specifically expressed in female and/or male germ cells, which has no consequences in both sexes. We discuss this notion of dispensability and the experiments that need to be carried out to definitively conclude that a gene is dispensable for a function

Diversity of X-inactivation initiation mechanisms in mammals

X-chromosome inactivation (XCI) in female mammals enables dosage compensation for X- linked gene products between the sexes. Since its discovery fifty years ago (Lyon, 1961), the developmental regulation of this process has been extensively investigated in mice (see Morey and Avner,2011; Augui et al, 2011; Gendrel and Heard, 2011 for reviews), but hardly at all in non-murine species. In mice, the X chromosome of paternal origin (Xp) is silenced during early embryogenesis due to imprinted expression of the regulatory RNA, Xist (X- inactive-specific-transcript). Paternal XCI initiates early in mice and is then reversed in the inner cell mass (ICM) of the blastocyst. Random inactivation of either paternal or maternal X then ensues in epiblast cells. We investigated the developmental regulation of XCI in rabbit and human embryos (Okamoto et al, 2011) and were able to show that in these mammals, Xist is not subject to imprinting and X inactivation begins much later than in the mouse. Furthermore, Xist is up-regulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the ICM. In rabbits, we showed that this triggers XCI on both X chromosomes, implying that the choice of which X chromosome will finally become inactive occurs downstream of Xist up-regulation, a situation that is totally different to the mouse, where choice occurs upstream of Xist RNA accumulation. In humans, on the other hand, XCI is not triggered, even by the blastocyst stage, despite the up-regulation of Xist. Xist is thus expressed, rather than repressed, in Oct4/Nanog positive ICM cells, in both non- murine species. In fact, the status of the two X chromosomes in the ICM is very different in rabbit, human and mouse embryos. In human ICM cells, the two X chromosomes seem to be active, while in the rabbit ICM, the two Xs are initially active (day 4) but then XCI initiates one day later (day 5) in the ICM. This is exactly the opposite situation to that found in the ICM of mouse blastocysts, where the paternal X is initially inactive and then becomes reactivated in the epiblast cells of the ICM. All these results demonstrate the remarkable diversity of X-inactivation initiation mechanisms and highlight major differences between mammals in the requirement for dosage compensation during early embryogenesis

DMRT1 is a Sex Determining Gene in Rabbits

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DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility

International audienceDMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1 −/− rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1 −/− ovaries did not undergo meiosis and folliculogenesis. XX DMRT1 −/− adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility

DMRT1 is Required For Sex Determination in Rabbits

International audienceThe gonad-specific transcription factor DMRT1 is considered as the Testis Determining Factor in many vertebrates. In mammals, SRY is the testis-determining gene, and according to the mouse species, DMRT1 has no longer function in testis determination since Dmrt1 disruptions only affect post-natal testis differentiation and fertility. Nevertheless, human patients with DMRT1 mutations exhibit 46, XY DSD with testicular dysgenesis, suggesting an earlier role for DMRT1 in non-rodent mammals. Thanks to CRISPR/Cas9 technology, our laboratory generated DMRT1 mutant rabbits. We showed that the absence of DMRT1 leads to early male-to-female sex reversal in XY homozygous mutants that developed ovaries. Indeed, when observed a few days after sex determination, XY DMRT1-/- gonads were devoid of testis cords structures and presented a typical ovarian organization with a thicker epithelium and ovigerous cords. Molecular studies confirmed the ovarian signature with an up-regulation of ovary-driving genes (RSPO1, WNT4, FOXL2, and CYP19A1) and downregulation of testis-specific genes (SOX9, SOX10, and DHH) in the developing XY DMRT1-/- gonads (bulk RNA-sequencing and RT-qPCR data). Interestingly, although some SOX9 protein remained detectable in a few somatic cells, it was not sufficient to trigger its target gene expression. Our data highlight the role of DMRT1 as a key player in sex determination in rabbits. DMRT1 is required for SRY action on SOX9 gene expression and may also be necessary for SOX9 protein function on its target genes acting as a pioneer factor. Our investigations are currently focused on characterizing DMRT1 direct targets in the rabbit gonads by ChIP-seq