CONICET | Buscador de Institutos y Recursos Humanos

Pronuclear microinjection and ICSI-mediated gene transfer (ICSI-mgt) are useful techniques to obtain transgenic animals. Nevertheless, a high frequency of mosaic expression was observed in embryos and offspring produced by these techniques. A possible explanation is that the transgen integrates in the embryo genome after the first cell divisions. Our main objective was to develop a new technique to generate transgenic bovine embryos, without mosaic expression and with high efficiency. We hypothesize that fertilizing MII oocytes with transgenic androgenetic haploid blastomeres (AHB) (from mosaic embryos), non-mosaic transgenic embryos are obtained. To this aim, in the first experiment we generated AHB enucleating in vitro matured MII oocytes, prior or after injecting with a single spermatozoon incubated with pCX-EGFP plasmid. These treatments were analyzed by Fisher test (P<0.05). The rate of cleavage of the androgenetic transgenic embryos enucleated prior and after ICSI-mgt was 35.1% (34/97) and 61.2% (71/116) respectively; (P<0.05). These embryos showed a EGFP expression of 11.8% (4/34) and 42.3% (30/71) (P<0.05) with 0% (0/34) and 9.9% (7/71) of non-mosaic expression. The haploid condition of the androgenetic embryos was confirmed by karyotype analyzes. After this first approach, we chose the procedure of enucleation after ICSI for successive experiments. In the second experiment, the haploid androgenetic embryos (4-16 cells) were disaggregated, and the AHB obtained were used to fertilize MII oocytes. Fertilization was carried out fusing a single AHB to a zona-free MII oocyte and then chemically activated. Presumptive zygotes were cultured in SOF medium in well of the well system. To confirm fertilization, single AHB produced with sexed Y spermatozoa and embryos generated with them, were checked by PCR using Y and X specific sequences primers. PCR analysis confirmed Y specific sequences in all the AHB, and XY specific sequences in each of the analyzed embryos. FISH analysis on blastocysts was performed with a specific probe for a Y chromosome sequence, confirming the sexed sperm genome in all blastocyst cells. Additionally, the Oct-4 (pluripotent marker gene) pattern expression was examined in the blastocysts, by immunocytochemistry with a confocal microscope. Blastocysts displayed a pattern of Oct-4 expression similar to IVF-embryos, indicating an efficient nuclear reprogramming. Finally, we fertilized MII oocytes with EGFP-AHB to produce transgenic bovine embryos without mosaic expression. The development reached 85.1% of cleavage and 9.0% of blastocysts (n=84). One hundred % of the embryos showed EGFP expression, with 90.1% of non-mosaic expression. In conclusion, our results proved that it is possible to use AHB for fertilization of MII oocyte, and that fertilization with transgenic AHB has demonstrated to be a high efficient technique for the generation of transgenic non-mosaic bovine embryos.

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