An Improved Enucleation Method of Bovine Somatic Cell Nuclear Transfer

Institute of Bio-Engineering, Northwest A & F University, Yangling 712100, China

More Information

Corresponding author: E-mail address: hs863@nwsuaf.edu.cn (Yong Zhang)

摘要

- /
- /
- /
Abstract: The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch²-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase ? stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls (87.4% vs. 64.4%, P < 0.05). Furthermore, centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.
- bovine /
- centrifugation /
- nuclear transfer /
- oocyte intraplasmic injection

HTML全文

参考文献(14)

[1]	Wilmut, I, Schnieke, et al. Viable offspring derived from fetal and adult mammalian cells Nature, 385 (1997),pp. 810-813
[2]	Guo, JT, An, et al. Cloned goats (Capra Hircus) from adult ear cells Sci in China, Series C., 45 (2002),pp. 260-267
[3]	Kubota, C Six cloned calves produced from adult fibroblast cells after long-term cultures Proc Natl Acad Sci USA, 97 (2000),p. 990
[4]	Tsunoda, Y, Shioda, et al. Differential sensitivity of mouse pronuclei and zytote cytoplasm to Hoechst staining and ultraviolet irradiation J Reprod Fertil, 82 (1988),pp. 173-178
[5]	Russell, DF, Ibez, et al. Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro Cloning and Stem Cells, 72 (2005),pp. 161-170
[6]	Smith, LC Membrane and intracellular effects of ultraviolet irradiation with Hoechst 33342 on bovine secondary oocytes matured in vitro J Reprod Fertil., 99 (1993),pp. 39-44
[7]	Tatham, BG, Dowsing, et al. Enucleation by centrifugation of in vitro-matured bovine oocytes for use in nuclear transfer Biology of Reproduction, 53 (1995),pp. 1088-1094
[8]	Silva, CP, Kommineni, et al. The first polar body does not predict accurately the location of the metaphase ? meiotic spindle in mammalian oocytes Fertil Steril., 71 (1999),pp. 719-721
[9]	Hardarson, TH, Lundin, et al. The position of the metaphase ? spindle cannot be predicted by the location of the first polar body in the human oocyte Hum Reprod., 15 (2000),pp. 1372-1376
[10]	Li, XF, An, et al. Effects of sources, serum starvation and preactivation of bovine donor cells on development of nuclear-transferred embryos in vitro Zoological Research, 24 (2003),pp. 35-38
[11]	Li, YQ, An, et al. Bovine somatic cell nuclear transplanation by intraplasmic injection Act Veterinaria et Zootechnica Sinica, 36 (2005),pp. 33-37
[12]	Takahashi, Y, First, et al. In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acid and vitamins Theriogenology, 37 (1992),pp. 963-978
[13]	Ebert, KM, Hammer, et al. A simple method for counting nuclei in the preimplantation mouse embryo. Experientia., 41 (1985),pp. 1207-1209
[14]	Chung, JT, Downey, et al. Reprod Fertil Dev., 13 (2001),pp. 383-388