The Mouse Genetics Engineering Center(Centre d'Ingénierie Génétique Murine, CIGM) is the Institut Pasteur’s transgenic mouse core facility. Since its creation in 2003, the CIGM aims to generate new models of transgenic mice by “classical” transgenesis, lentivirus-mediated transgenesis,and also by targeted transgenesisafter homologous recombination in Embryonic Stem (ES) cells. Preferentially open to theInstitut Pasteurscientific community, the services of CIGM are also available to outside customers interested in new transgenic,knock-down, knock-out(KO) orknock-in(KI) mouse models. The core facility also provides expert advice regarding the design of gene constructs suitable for transgenesis and homologous recombination experiments.

The classical transgenesisservice involves the microinjection of the gene of interest into the pronucleus of fertilized eggs in order to generate transgenic mice expressing the transgene. The microinjected transgenes include DNA linear fragments(<25kb) as well as larger DNAs from bacterial or yeast artificial chromosomes (BACs, YACs). Moreover, we also perform lentiviral transgenesis experiences, consisting in subzonal microinjections of the lentiviral construct. The genetic backgrounds proposed for classical transgenesis include hybrid mouse lines, such as B6SJLF1, B6D2F1, B6CBAF1 or B6BALB/cF1, as well as pure genetic backgrounds, such as C57BL/6, BALB/c or FVB. In the period 2009-2010, we have successfully created an increasing number of BAC-mediated transgenic mouse lines using the most difficult genetic background C57BL/6. Interestingly, in 2010 we also realised first experiences of ES-cell mediated BAC transgenesis.

The homologous recombination technique in ES cellsand the subsequent microinjection of these modified ES cells into mouse blastocysts allow the generation of germ-line chimeras for targeted transgenesisin the endogenous gene. Until 2009, we generated more than 40 heterozygous/homozygous mice lines corresponding to new KOor KImouse models, always using “classical” 129-derived ES cell lines (i.e. 129Sv, 129Ola, AB1, TC1, CK35RosaERT2).

Interestingly, in 2009 we successfully started the culture of different C57BL/6-derived ES cells. Thanks to the availability of a good germ-line competent wild-typeB6 ES cell line, we performed in 2010 the first successful homologous recombination experiences with isogenic C57BL/6 targeting vectors, in order to directly generate heterozygous/homozygous mice in this pure inbred genetic background (Figure 1). Obviating the need for backcrossing to obtain the desired defined C57BL/6 background constitutes a faster route to final phenotype analysis and also reduces husbandry efforts and mouse housing that are expensive in labour, materials and space.

Finally, since its creation, CIGM keeps interacting with a steadily increasing list of research groups made up of 34 Units within the Institut Pasteur(belonging to 7 out of the 10 Departments), and other Research Institutions in France (CNRS UnitsUMR 7622-Paris 6, UMR 7592-Institut Jacques Monod-Paris, UMR 146-Institut Curie-Orsay, UMR 6061-Rennes, UPR 1142-Montpellier, INSERM Units U470-Lariboisière, U787-Pitié-Salpétrière, U813-Necker, U872-Cordeliers and E9935-Robert Debré-Paris, and U522-Pontchaillou-Rennes, UMR 910 La Timone-Marseille, UMR 955-INRA-ENVA-Alfort, the Institut National de la Transfusion Sanguine-Paris) as well as in foreign countries (Université Libre de Bruxelles- Belgium, Universitat de Barcelona-Spain, Institute Fondazione Rita Levi-Montalcini Roma-Italy).

Keywords: Mouse embryos, Transgenesis, Gene Targeting, Homologous Recombination, Embryonic Stem (ES) cells, Microinjection, DNA transgene, BAC, Lentivirus

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