The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda-rabbit interspecies nuclear transfer.
Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda-rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda-rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p > 0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda-rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.
Jiang
Man-Xi
MX
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Yang
Cai-Xia
CX
Zhang
Li-Sheng
LS
Zheng
Yue-Liang
YL
Liu
Shu-Zhen
SZ
Sun
Qing-Yuan
QY
Chen
Da-Yuan
DY
D000818:Animals; D000001:Calcimycin; D002467:Cell Nucleus / Q000187:drug effects; D019976:Cloning, Organism / Q000379:methods*; D003572:Cytochalasins; D003703:Demecolcine / Q000494:pharmacology; D047108:Embryonic Development / Q000502:physiology*; D015759:Ionomycin; D008297:Male; D008845:Microinjections / Q000379:methods; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000187:drug effects; D011817:Rabbits; D014183:Transplantation, Heterologous; D001503:Ursidae / Q000196:embryology*
D003572:Cytochalasins; D000001:Calcimycin; D015759:Ionomycin; D003703:Demecolcine