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Kif2a Conditional Knock-In Mouse: A Flexible Model To Study Cortical Malformations And Epilepsy

Reference : PhD Jamel CHELLY

Publication de l'offre : 5 avril 2016

Genetic studies of our group and others demonstrated that mutations in genes encoding for tubulins, centrosomal and MT-dependent motor proteins such as TUBG1, DYNC1H1, KIF5C and KIF2A are implicated in a large spectrum of malformations of cortical development (MCD) associated with intellectual disability and epilepsy ( Poirier K et al., et al. (2013). Nat. Genet. 45, 639–647).

KIF2A is a member of the kinesin-13 family, which differs from conventional kinesins as rather than regulating cargos transport along microtubules (MTs); it is involved in MT dynamics ( Hirokawa N et al., (2010). Neuron 68, 610–638). To further characterize the effects of KIF2A missense mutations in pathophysiological mechanisms that mimic mutations-related disorders, we initiated, in collaboration with the Institut Clinique de la Souris (ICS), the development of a conditional knock in Kif2a mouse model expressing the mutation c.961C>G, p.His321Asp previously detected in a patient with pachygyria and microcephaly. Though constitutive knock in mouse models are
a straightforward and widely used approach, generation of conditional knock in models remains highly challenging. This can be achieved through specific engineering of the mutant allele and the Cre-dependent one-way genetic switch (FLEx switch) to generate the knock-in model (Schnütgen F et al., (2003). Nat. Biotechnol. 21, 562–565).

So far, we have applied this innovative strategy and provided a proof of concept using engineered recombinant embryonic stem (ES) clones. Indeed, functionality of FLEx system, as well as the efficient one-way reversion leading to the expression of the mutant KIF2A upon transfection with a Cre-recombinase expressing vector, was validated by RT-PCR and sequencing of the cDNA fragment corresponding to the exon of interest, and
analysis of KIF2A protein by immunofluorescence. Following validation of the system in ES cells, chimera mice were already generated and used to generate heterozygous animal models Kif2aFLEx-mut/+. Heterozygous animals will then be crossed with transgenic mice expressing either Cre-recombinase under the control of specific promoters or tamoxiflen-inducuble Cre-recombinase.

The PhD project will be focused on the investigation the mouse model conditionally expressing mutant KIF2A. It will include study of the consequences of the expression of KIF2A mutant on: (i) behaviour and learning performances, and susceptibility to epilepsy, (ii) layering pattern of the cortex during development, (iii) assessment of the consequences of the mutation on mitotic and cell cycle processes, as well as post-mitotic neuronal polarization, migration and early stages of differentiation, (iv) real-time live imaging on cultured brain slices and cultures of MGE explants to better dissect KIF2A mutant consequences on radial migration and tangential interneuron dynamics respectively.

Furthermore, to study the involvement of different cell types in the migrating process, and assess the possibility to generate focal lesions, we will rely on in utero electroporation of the conditional Kif2a mouse (FLExmut/+). In utero electroporation of vectors expressing Cre-recombinase under the
control of cell type specific promoters will enable us to selectively study the effect of mutant KIF2A expression in a restricted population of neuronal cells and its consequences on the organization and function of the cortex. In addition to its potential to generate an animal model for FCD and epilepsy, the generation of the conditional knock in Kif2a mice will provide insights for a deeper understanding of KIF2A function and role in cortical development, as well as pathophysiological mechanisms underlying KIF2A-related MCD.

- COMPETENCES SOUHAITEES : Neuroscience et Neuro-developpement, Biologie moléculaire et cellulaire, Imagerie cellulaire et imagerie en temps réel, Expérimentation animal

- EXPERTISES QUI SERONT ACQUISES AU COURS DE LA FORMATION : Neuro-développement, Corticogenèse normale et pathologique knock in conditionnel, Exploration de processus neuro-développementaux dans des modèles animaux,
Electroporation in utero, Rôle des kinésines dans le développement (KIF2A en particulier)

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Date limite de candidature : 31 décembre 2016

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