> Stages de M2 > Liste des stages proposés pour l’année 2015-2016 > Implication of nuclear pores constituents in cell cycle and (...)

Implication of nuclear pores constituents in cell cycle and development.

proposé par Valérie DOYE, Institut Jacques Monod, UMR7592, CNRS, Université Paris Diderot, 15 rue Hélène Brion, 75013 Paris

Projet de stage :

The specific aim of this project will be to characterize the functions of Y-Nups other that Nup133 during cell cycle progression and cell differentiation. Therefore, a CRISPR/Cas9-based approach will be developed in mESCs. Indeed, this model system, which is now well established in our team, enables to perform functional studies in non-cancer cells, and to characterize protein functions in cell division and differentiation in both pluripotent stem cells and in their in vitro differentiated derivatives. At the structural level, Y-Nups are characterized by a-solenoid and/or ß-propeller domains. In view of the key function of the ß-propeller domain of Nup133 in mESC differentiation (our unpublished data), the initial screen will focus on the inactivation in mESCs of the 4 other ß-propeller containing Y-Nups. If these proteins are not required for ES cell viability, the in vitro differentiation properties of these KO cell lines will be characterized using monolayer neural and mesoendodermal differentiation protocols followed by viability assays, morphological analyses, immunofluorescence and qRT-PCR studies (well-established procedures in the team). If for some Y-Nups, only heterozygous lines are obtained this will indicate that the corresponding Y-Nup is essential for viability of undifferentiated ES cells. In that case, and depending on the outcome of the other Y-Nups inactivation, a CRISPR/Cas9 strategy will be used to introduce defined mutations in the ß-propeller. In a longer term, these studies, combined with the functional characterization of direct partners of these Y-Nups should help to elucidate to which extent Y-Nups-dependent coordinated regulations of cell proliferation, division, and differentiation may contribute to the proper development of multicellular organisms.

Techniques mises en œuvre par le stagiaire :
- cell culture and differentiation (mainly mouse embryonic stem, mESCs, and neuronal differentiation)
- CRISPR/Cas9 mediated mutagenesis in mESCs
- phenotypic analyses : immunofluorescence, FACS, qRT-PCR, western blot, ..

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