Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer |
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Authors: | XuDong Guo DongShan Yang XuDong Ao Xia Wu GuangPeng Li LingLing Wang Ming-Tao Bao Lian Xue ShorGan Bou |
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Institution: | (1) Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot, 010021, China;(2) Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510663, China |
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Abstract: | In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression
vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin
resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear
transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods,
one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were
respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa
and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups
(P>0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which
the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were
17 embryos out of 31 strongly expressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to
identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neo
r
genes were correctly expressed indicating that transgenic somatic cell lines and positive transgenic embryos were obtained;
(ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only
partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization,
transgenic cashmere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which
was sufficient for production of transgenic cashmere goats.
Supported by the National High Technology Research and Development Program of China (Grant No. 2002AA242061) and Natural Science
Foundation of Inner Mongolia (Grant No. 200607010405) |
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Keywords: | cashmere goat insulin like growth factor 1 hair follicle fetal fibroblasts cells transgene somatic cell nuclear transfer |
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