Nuclear transfer in loach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the loach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 °C in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days. It was found that gastrula cells incubated at 4 °C had the same totipotence as blastula cells. The optimal UV dosage for inactivation of the oocyte chromatin was 180–240 mJ cm^?2. Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.