Modifications of chemically induced-enucleated nuclear transfer technique by reverse-order nuclear transfer in mouse

To improve the developmental potential of somatic cell cloned embryos derived from demecolcine (DC) induced-enucleated nuclear transfer (INT), we modified the INT procedures by transferring donor nuclei into recipient cytoplasts prior to the induced enucleation of the recipient cytoplasts, and we called this modified INT technique as reverse-order and induced-enucleated nuclear transfer (RINT). Standard nuclear transfer (SNT) and INT were performed as controls. The dynamic changes of maternal and transferred donor nuclei in the RINT oocytes were monitored to evaluate the feasibility of this new nuclear transfer (NT) technique by timed immunofluorescence. Timed immunofluorescence showed that RINT is feasible because none of the transferred donor nuclei were expelled with the second polar body (Pb) in the RINT oocytes, while 42.2% of the oocytes showed extrusion of all maternal chromosome and spindles with the second Pb at 60 min after activation and DC treatment. Although there was no difference in cleavage rate (86.6% vs. 82.1%), the rates of successful enucleation and blastocyst formation were significantly increased in RINT compared with INT (44.1% vs. 27.5% and 43.3% vs. 12.8%, respectively; p < 0.01). Compared with SNT, there was no difference in cleavage rate (86.6% vs. 78.4%), but the blastocyst developmental rate was significantly increased in the RINT group (43.3% vs. 25.3%; p < 0.01). Blastocysts derived from RINT had a higher total cell number than those from SNT (45.1 ± 3 vs. 37.6 ± 4; p < 0.05). Our results provide evidence that RINT is feasible and may provide a more efficient and simple method for NT than INT.