Cloned goats (Capra hircus) from adult ear cells

Abstract

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the M II chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5 mumol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells precooled at 4 degrees C for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.

Effects of donor sex and different cell treatments on development of yak–bovine interspecies nuclear transfer embryos

“…Guo et al (2002) found that there was no effect on embryo development by keeping donor cells at 4 C for 1 week. We used frozenthawed and fresh cells as donors to test the effect of freezing on interspecies NT, and found that cleavage rates differed between the two groups, but developmental rate and embryo quality did not.…”

In vitro developmental competence of pig nuclear transferred embryos: effects of GFP transfection, refrigeration, cell cycle synchronization and shapes of donor cells

“…Currently, there are several ways for preparing somatic cells for NT, including fresh isolation of cells from tissues (Wakayama et al, 1998), fresh detachment of cells from cultured cell lines (Cibelli et al, 1998), thawing of cryopreserved cells (Lai et al, 2002a), and freezing of cells stored at 4 • C (Guo et al, 2002;Liu et al, 2001). It was reported that refrigerated cells can support the development of nuclear transferred bovine embryos to blastocysts at a comparable rate to controls (Liu et al, 2001).…”

Contrasting effects of G1.2/G2.2 and SOF1/SOF2 embryo culture media on pre- and post-implantation development of non-transgenic and transgenic cloned goat embryos

“…Different culture media have been used for in-vitro culture of the reconstructed goat embryos, including Charles Rosenkrans medium (CR1) (Chen et al, 2007;Guo et al, 2002;Lan et al, 2006), tissue culture medium (TCM)-199 (Baguisi et al, 1999) and synthetic oviductal fluid (SOF) (Melican et al, 2005).…”