RESUMO
Previous methods used for nuclear transplantation were further investigated to develop a method that was both easy to carryout and did not require any special apparatus, such as Piezoimpact or Spindle-View. Following the puncture of zona pellucida with two holes by injection pipette that contained donor nuclei or cells, the injection pipette was pulled back to the perivitelline space while the negative pressure was increased in the holding pipette until the polar body and karyoplasm were wiped off completely. Then a reconstructed embryo was completed by the direct injection of the donor nucleus or cell without pulling out the injection pipette. 200 oocytes were manipulated using this method and it cost about 40 seconds with nucleus injection and about 30 seconds with cell injection to complete a reconstructed embryo. The success rates were 62.6% and 86. 0%, respectively, and enucleation rate was about 73.3% validated by Hoechst 33342. Using this method, the nucleus was completely eliminated and another was injected using the microscope and micromanipulator. Moreover, the efficiency of nuclear transplantation and survival rate of reconstructed embryos were greatly improved. Furthermore, it is very easy to manipulate and popularize in practice.
