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Abstract :
[en] We have previously generated genetically engineered (GE) mice by pronuclear injection of a bacteri-al artificial chromosome (BAC) expressing the complete Bos taurus antiviral Mx system. It turned out that these mice display exceptional anti-viral activities against lethal infections with influenza A viruses. As swine are highly susceptible to infection with both avian and human influenza A viruses, new potentially life-threatening variants can appear by reassortment in the pig lung. For testing the hypothesis that ex-pressing a Mx isoform endowed with strong antiinfluenza activity would reduce the risk of disseminating reassortants, we aim to generate bovine Mx1-expressing pigs. The first step to complete such initiative was to establish (from scratch) a suite of technologies generating ready-for-transfer GE porcine embryos from BAC DNA, frozen sperm, and ovaries collected in local slaughter plants. Here, we report the effi-ciency of our current process for generating such embryos after complete in vitro production in defined media, successively including (i) in vitro maturation of oocytes, (ii) BAC-loading of sperm, (iii) in vitro fertilization and (iv) in vitro development of zygotes until the blastocyst stage. Fertilization and genetic modification were obtained via sperm-mediated gene transfer (Watanabe et al., 2012). About 2.6% of slaughterhouse-derived oocytes gave blastocysts. This rate is relatively low at first sight but, contrary to other teams, we used defined media only, which guarantees the reproducibility and biosafety of the process over time. Further, 64% of these blastocysts had incorporated at least a fragment of the BAC, of which about two thirds had incorporated the complete sequence of the BAC. Generating ready-for-transfer GE pig embryos is thus feasible using the suite of technologies we currently have in hands. All blastocysts produced since several months are now cryopreserv ed by vitrification for a future transfer in surrogate sows.
