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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

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

The DDK inbred strain of mice carries a conditional lethal 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) have 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. In addition, we have shown that the disruption of these genes (by targeted mutagenesis in ES cells) results in early embryonic lethality, thus demonstrating their essential role in embryonic development.

2. Functional analysis of HNF1b and Trapa

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, we have demonstrated that HNF1b inactivation in the primitive gut result in defects in the development of this organ. We are now developing a molecular analysis of these defects by probing in the first place various molecular markers already known to be implicated in these aspects of differentiation and morphogenesis.

Functional analysis of an insertion mutation resulting in defective cardiac morphogenesis (J. Barra)

Using a gene trap approach, we obtained an insertion mutation resulting in the homozygous state in embryonic lethality after mid-gestation. We showed that the gene trap vector was inserted in the TRAPa gene which codes for a subunit of a protein complex associated with the translocon. Analysis of the phenotype of mutant embryos demonstrated a defective cardiac morphogenesis, including a cardiac hypertrophy and a septation defect inducing a gap between ventricles. We are currently examining the possible implication, either direct or indirect, of the cardiac neural crest cells in the observed phenotype.

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 targeted. 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 restoration 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 significant 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.

Keywords: Mouse embryonic development, ES cells, HNF1b, Trapa, nucleocytoplasmic interactions, gene targeting, I-SceI



  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)

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

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

ARTUS Jérôme, PhD student

CORMIER Sarah, postdoc

COUMAILLEAU Franck, PhD student

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)


Activity Reports 2002 - Institut Pasteur
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