Sexing Of Mouse Morulae Vitrified By OPS Method With Diplex, Nested PCR

In this experiment, two aspects were mainly focused: the measuration of the sensitivity of diplex, nested-PCR for sexing embryos of different biopsy amount, and sexing mouse embryo vitrified by OPS method using diplex and nested-PCR, which developed the embryo sexing technology system of the mammalian animal, especially the sexing of embryo freezed, and provided valuable parameters for higher efficiency in practice of the embryo sexing, especially embryo freezed.The sensitivity of diplex, nested-PCR was measured in order to provide technological support for molecular technologies, such as PGD and animal embryo sexing. The SRY/ZFX diplex and nested-PCR embryo sexing technological system specific for mouse was established. The sensitivity of diplex and nested-PCR was measured according to the efficient detection rate of amplifying blastomeres varying from 1 to 5 or above, and five groups were made accordingly. In the first group, the efficient detection rate, contamination rate and failure rate was 85 % (34/40 ), 7.5 % (3/40) and 7.5% (3/40) , respectively. With the increase of blastomeres, the contamination rate and failure rate both decreased accordingly, contrast to the efficient detection rate. In the fifth group, the efficient detection rate and failure rate reached 100%, 0, respectively. The efficient detection rate and failure rate of the first group were significantly different with those of the other groups (p<0.05), but they showed no difference (p>0.05) between the other groups. Conclusion: Diplex and nested-PCR can detect efficiently about 12 pg genome DNA, and the target fragment is of single copy.Morulae vitrified by OPS and fresh morulae were sexed with the above system individually. The morulae were biospied by hand, and vitrified by two-step method (10%E+10%D,30s→EDFS30,25s →LN₂),thawed by one-step method (0.5mol/L Sucrose solution,5min→mPBS→CO₂ Incubator). 324 morulae vitrified by OPS, 256 fresh morulae were sexed, respectively. 301 of 324, 232 of 256 were transferred into 22, 16 recipients. 28, 21 fetuses were obtained from 11 of 22 recipients, 7 of 16 repcipients individually, and both were in accordance with the sex predicted.