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  Director : BABINET Charles (chbabi@pasteur.fr)


  abstract

 

Our laboratory is studying mouse embryonic development. Three lines of research are conducted : 1) We are studying the role of nucleocytoplasmic interactions in mouse preimplantation development. 2) We study the function of different genes in development via gene targeting approaches and we use a strategy of gene trapping to identify developmentally important genes. 3) We develop strategies to improve the efficiency of gene targeting.



  report

cale

1. Cloning of the Ovum mutant mutation (Om) which induces embryonic death around the blastocyst stage and entails a parental effect (P. Baldacci, M. Cohen-Tannoudji)

The DDK inbred strain of mice carries a conditional letal mutation (we call the death of the embryos: "DDK syndrome") which is manifested in outcrosses and depends on the direction of the cross. We showed that the DDK syndrome entails an interaction between a DDK cytoplasmic product contained in the zygote and the alien paternal genome. We had mapped the Om locus and have now established its physical map by isolating a contig of BACs which encompasses the genetic region containing Om. Exon trapping, cDNA selection and sequencing (in collaboration with the Génoscope, Evry, France) has allowed us to identify ca. fifty genes. Among those, two of them, are privileged candidates to be involved in the DDK syndrome: indeed they are expressed in the oocyte and the testis; furthermore, they exhibit a difference in their open reading frame which is unique to DDK. We now develop functional approaches which should allow us to demonstrate that these mutations are indeed directly involved in the DDK syndrome.

2. Targeted mutagenesis to analyse gene function in vivo and search for developmental genes by a gene-trap approach

The function of HNF1b (J. Barra in collaboration with the group of Dr. M. Yaniv, Pasteur Institute)

We had shown that embryos homozygous for a null mutation in HNF1b gene die around day 7 of gestation, due to a defect in visceral endoderm differentiation. This year, we addressed the role of HNF1b in organogenesis, using a strategy of conditional mutagenesis. We have shown that specific disruption of HNF1b in hepatocytes and bile ducts prevents the formation of intrahepatic bile ducts; furthermore severe abnormalities of the gallbladder are observed. We also identified HNF1b targets involved in bile sensing and fatty acid oxidation. We extended our observations to the effects of HNF1b specific inactivation in epiblast which results in defects in the morphogenesis of liver, pancreas and kidney. Furthermore, preliminary results indicate that HNF1b inactivation in the primitive gut result in defects in the development of the gut.

- Gene trap in ES cells (J. Barra)

This approach is aimed at identifying developmentally important genes, via the creation of insertion mutations. The latter are generated via the random insertion in ES cells of a "trap" vector comprising a reporter gene (coding for a fusion protein between b-galactosidase and a protein confering resistance to an antibiotics), without promoter/regulatory sequences. Thus the reporter gene is expressed only if inserted functionally in a gene active in ES cells. We have pursued this year the analysis of an insertional mutation in the TRAPa gene. Indeed, we demonstrated that the embryos homozygous for the trap insertion die starting at day E14 of embryonic development. The embryos exhibit a severe defect in cardiac morphogenesis: the heart is hypertrophic and the septation is completely abnormal resulting in the absence of separation between ventricles.

3. An approach to improve the efficiency of gene targeting (M. Cohen-Tannoudji)

We have recently developped a strategy for mouse germline modifications based on the stimulation of endogenous DNA repair processes. This approach allows to enhance considerably the targeting efficiency at a given locus. It consists in the introduction of recognition site for the meganuclease I-SceI into the locus to be targetted. I-SceI induces a double strand break in the locus which stimulates homologous recombination with an incoming repair matrix. Our aim is now to: i) to improve the targeting methods by introducing targeted modifications directly into the zygote, thus avoiding the use of ES cells. ii) Create genetic modifications in somatic cells. iii) Identify permissive sites for the targeted expression of genes involved in human pathologies or of therapeutic agents.

Recently we have generated a reporter gene comprising the coding sequences of LacZ separated by an I-SceI site. The reporter gene is not functional, due to the presence of LacZ duplicated regions surrounding the I-SceI site. Recombination can then be estimated by the restauration of LacZ activity which is easily monitored. Thus we have coinjected the reporter gene with I-SceI protein into the pronucleus of fertilized egg and could observe b-galactosidase expression in a significative proportion of the embryos. We now wish to extend this result which demonstrates extra-chromosomal recombination to the case of a reporter gene integrated into the genome. To that end, transgenic mice obtained via ES cells and containing this new reporter gene, are being generated. We will then inject I-SceI into the zygotes bearing the reporter transgene; in this way we will be able to evaluate the possibility of enhancing intra-chromosomal recombination in ovo.

A core facility for the generation of transgenic mice (S. Rudinger)

Our laboratory is in charge of a transgenic facility which generates transgenic mice for the groups of the Pasteur. Institute.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

FLEURANCE Isabelle (ifleur@pasteur.fr)

BABINET Charles, CNRS and IP, (Researcher, chbabi@pasteur.fr)

BALDACCI Patricia, IP, (Researcher, baldacci@pasteur.fr)

BARRA Jacqueline, IP, (Researcher, barraja@pasteur.fr)

COHEN-TANNOUDJI Michel, CNRS, (Researcher, m-cohen@pasteur.fr)

ARTUS Jérôme, PhD student

COUMAILLEAU Franck, PhD student

LE BRAS Stéphanie, PhD student

PEYRON Fabienne, Engineer

KRESS Chantal (Engineer, kerssova@pasteur.fr)

MESBAH Karim (Technician, mesbahk@pasteur.fr)

RUDINGER Stéphanie (Technician, rudinger@pasteur.fr)

VANDORMAEL-POURNIN Sandrine (Technician, drinette@pasteur.fr)


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