Reconstruction of <i style="">in vitro</i> matured goat oocytes by using synchronized skin fibroblast cells as donor nuclei

Abstract

Cloning of embryos by nuclear transplantation has been developed in several species using foetal fibroblasts as donor cells. In the present study, the developmental potential of foetal fibroblasts, arrested in metaphase stage using cytochalasin-B, was evaluated using nuclear transfer. Studies were undertaken to find out suitable stage of the cell cycle of goatskin fibroblast cells with enucleated <i style="">in vitro</i> matured goat oocytes and their capability of development of embryo. Skin cells were isolated from fetus as well as from adult skin and cultured to monolayer using RPMI-1640 media with 10% FCS in 5% CO₂ incubator at 38.5º C ± 1º C with 95% humidity. Immature oocytes were matured <i style="">in vitro</i> for 22-24 hrs and enucleated using a Leitz micromanipulator. The cytochalasin-B blocked synchronized cells were used as donor cells for transferring into the perivitelline space of the enucleated oocyte and electrofused with constant AC pulse at 7-10 volts, while DC pulses at 180-400 volts were applied for 5-20 <img src='/image/spc_char/micro.gif'> sec followed by culturing in CO₂ incubator to observe proper electrofusion and cleavage. The embryos after fusion were activated with cytochalasin-B for 2-4 hrs and then transferred to IVC media. Foetal fibroblast became confluence within 2 days whereas adult fibroblast cells took 7-8 days to become confluence and, by 1-2 hr of cytochalasin-B treatment, around 85% foetal fibroblast cells arrested in the metaphase stage and 4.3% reconstructed embryos reached to 2-cell and morula stage in each category. Thus, 3-5 passaged foetal fibroblast cells synchronized by cytochalasin-B and around 300 volts would be better for electrofusion for reconstruction of goat oocytes.