[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.


CONCLUSIONS
The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.
Shen
Xin-ming
XM
Institute of Cancer Research, Southern Medical University, Guangzhou 510515, China. xmshen2002@hotmail.com
Qiao
Gui-lin
GL
Jiang
Pei-zhou
PZ
Li
Xin
X
Huang
Hua
H
Yao
Kai-tai
KT
D000818:Animals; D002478:Cells, Cultured; D004624:Embryo Transfer; D047108:Embryonic Development; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology*