63 OOCYTE GENOME CLONING USED IN TRANSGENIC BOVINE EMBRYO PRODUCTION

Abstract

Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogenetic haploid blastomeres (PHB) from these embryos should be considered as a clone of the original gamete. Our main objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Moreover, we generated biparental homogeneous transgene-expressing embryos using PHB that express a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation in 5 μM ionomycin for 4 min followed by culture in SOF for 3 h to permit second polar body extrusion and subsequently treated with 1.9 mM DMAP for 3 h. To generate transgene-expressing PHB, parthenogenetic embryos were injected 3 h post-activation with pCX-EGFP–liposome complexes. These treatments were analysed by Fisher test (P < 0.05). The cleavage rate of the haploid and diploid parthenogenetic embryos control was 87.3% (103/118) and 88.5% (108/122) respectively; (P > 0.05). When the haploid activated oocytes were injected with pCX-EGFP–liposome complexes, the cleavage rate was 84.4% (54/64). These embryos showed 54.7% (35/54) of EGFP expression. In the second experiment, haploid parthenogenetic embryos (4 to 16 cells) were disaggregated and the PHB obtained were fused with a zona-free haploid male hemizygotes in order to reconstruct biparental embryos. Haploid male hemizygotes were generated by fertilization of enucleated oocytes. The haploid condition of parthenogenetic embryos and male hemizygotes was confirmed by karyotype analyzes. Reconstructed embryos were cultured in SOF medium in well of well system in 5% O₂, 5% CO₂, and 90% N₂ at 39°C. The development of the reconstructed embryos expressing or not expressing EGFP reached 78.4% (29/37) and 61.1% (80/131) of cleavage; and 10.8% (4/37) and 8.4% (11/131) of blastocysts, respectively; (P > 0.05). EGFP expression was observed in 100% of the reconstructed embryos, with 96.6% (28/29) of homogenic expression. The biparental IVF control showed 64.4% of cleavage and 22.5% of blastocysts (n = 98). The mean cell number of blastocysts were significantly different among the reconstructed biparental embryos (mean = 72.8) and the biparental IVF-embryos control (mean = 97.0). Additionally, the Oct-4 pattern expression was examined in the blastocysts, by inmunocytochemistry. Blastocysts displayed a pattern of Oct-4 expression similar to IVF-blastocyst control. Finally, biparental reconstructed blastocysts (n = 2) were transferred in recipient cows obtaining 1 pregnancy detected at 2 months after embryo transfer. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that it is an efficient technique to generate homogeneous transgene-expressing embryos. Haploid oocyte genome replication would have high potential in livestock production, as it increases the number of embryos produced from a single oocyte, and allows the selection of favourable genetic characteristics before biparental embryo reconstruction.