Shen Xin-ming, Qiao Gui-lin, Jiang Pei-zhou, Li Xin, Huang Hua, Yao Kai-tai
Institute of Cancer Research, Southern Medical University, Guangzhou 510515, China.
Di Yi Jun Yi Da Xue Xue Bao. 2005 Jun;25(6):613-8.
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.
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.
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.
The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.
建立一种快速、简便、高效且微创的核移植方法，用于研究体外构建的以卵丘细胞核重构的小鼠胚胎的早期发育。
使用尖锐的去核针，在大鼠卵母细胞的透明带上作一个约25毫米长的切口，通过该切口移除第一极体，并通过轻轻挤压和真空抽吸卵母细胞，将中期II染色体 - 纺锤体复合物连同最少体积的细胞质一起吸出。将直径为10 - 12毫米的C57BL/6j小鼠的卵丘细胞核插入去核卵母细胞的卵周隙。通过电融合诱导供体 - 受体对融合，并观察核形成。将重构卵母细胞在体外培养72小时后，观察2 - 细胞、4 - 8细胞和桑椹胚阶段胚胎的发育情况。
改良的核移植方法能够一步从卵母细胞中完整移除细胞核，通过紫外光下对移除细胞核进行Hoechst 33342染色，证实了细胞核完全移除的可靠性。去核过程平均耗时15秒，去核卵母细胞的存活率达到95%。将卵丘细胞核插入去核卵母细胞透明带的成功率为76.7%，核移植后的重构卵母细胞中有62.2%发生原核形成。在CZB培养基中对重构卵母细胞进行72小时体外培养后，发育到2 - 细胞、4 - 8细胞和桑椹胚（超过16个细胞）阶段的胚胎百分比分别为57.5%、39.1%和27.6%。从重构胚胎的DNA中扩增微卫星序列（D7Mit22和D4Mit204）以鉴定其来源，结果证明其来源于C57BL/6j小鼠。
改良的核移植方法简单、微创、高效且可行，可用于用体细胞核重构小鼠胚胎。
