改进的核移植方法完成小鼠卵丘细胞重组胚的早期发育

目的建立一种快速、简便、有效且损伤小的核移植方法，研究来源于小鼠体细胞重组胚的早期发育。方法用尖锐的去核针在MⅡ期卵母细胞透明带上切开一个25mm左右的小口，首先将第一极体挑出．再轻轻地挤压和负压吸引卵母细胞，去除包含有MⅡ期染色体．纺锤体复合体的最少量细胞质。随后，将直径为10～12mm的C57BL／6j小鼠卵丘细胞核注射到去核卵母细胞透明带下，构建供体核．卵母细胞复合体。用电融合的方法诱导复合体融合．并对重组胚激活。体外培养72h．对重组胚发育到2细胞、4～8细胞和桑椹胚期等各阶段进行观察和计数。结果完成一个MⅡ期卵母细胞去核的平均时间为15s；Hoechst 33342染色证明可以完全去除MⅡ期卵母细胞细胞核；去核成功率为95．5％，注核成功率为76．7％，供体核．卵母细胞复合体融合和重组胚原核形成率分别为68.2％和62.2％；重组胚体外培养72h内发育到2细胞、4-8细胞和桑椹胚期的比率分别为57．5％、39．1％和27．6％；用2个微卫星位点D7Mit22和D4Mit204引物，扩增不同发育阶段重组胚的DNA片段，表明重组胚来源于供体卵丘细胞。结论应用改进的核移植方法，不仅可以有效地去除卵母细胞细胞核，去核成功率高，而且操作简便，去核迅速，对卵母细胞损伤小．是研究小鼠体细胞核移植的一种简便可行的新方法。

A modified method of nuclear transfer for investigating early development of mouse embryos reconstructed with cumulus cell nuclei]

OBJECTIVE: To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. METHODS: With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS: The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95%. The success rate of 76.7% was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2% of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION: The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.