Author/Authors :
Zheng, Xi-bang Northwest University of Agriculture and Forestry - Animal Medicine College - Shaanxi Branch of National Stem Cell Engineering and Technology Center, China , Zheng, Xi-bang Guangxi University - College of Animal Science and Technology - Department of Animal Medicine, China , Yun, Yan Northwest University of Agriculture and Forestry - Animal Medicine College - Shaanxi Branch of National Stem Cell Engineering and Technology Center, China , Hu, Yong-ce Northwest University of Agriculture and Forestry - Animal Medicine College - Shaanxi Branch of National Stem Cell Engineering and Technology Center, China , Li, Yong Ningxia University - College of Life Science, China , Wang, Hua-yan Northwest University of Agriculture and Forestry - Animal Medicine College - Shaanxi Branch of National Stem Cell Engineering and Technology Center, China , Ma, Xiao-ling Northwest University of Agriculture and Forestry - Animal Medicine College - Shaanxi Branch of National Stem Cell Engineering and Technology Center, China , Sui, Jin-qiang Northwest University of Agriculture and Forestry - Animal Medicine College - Shaanxi Branch of National Stem Cell Engineering and Technology Center, China , Lei, An-min Northwest University of Agriculture and Forestry - Animal Medicine College - Shaanxi Branch of National Stem Cell Engineering and Technology Center, China , Dou, Zhong-ying Northwest University of Agriculture and Forestry - Animal Medicine College - Shaanxi Branch of National Stem Cell Engineering and Technology Center, China
Abstract :
The purpose of this study was to establish Nanog-expressing cell lines that can be used as donor cells to construct transgenic cloned embryos, and to investigate their in vitro development competence. By reverse transcription-polymerase chain reaction (RT-PCR), the cDNA of Nanog gene was cloned from fetal bovine primordial genital ridge tissues. The gene was inserted into PMD18-T vector using recombination techniques and then subcloned into vector pEGFP-C1. After confirmation by restrictive endonuclease digestion and sequencing, the recombinant plasmid pEGFP-Nanog was transfected into skin fibroblast cells. A stable transfected cell line was successfully established after two months of selection with neomycine (G418). Fluorescence microscopy, RT-PCR, and Western Blotting assays indicated that Nanog mRNA and EGFP-Nanog fusion protein were expressed in these cells. The EGFP-Nanog expressing fibroblast cells and the intact fibroblast cells (BEF422) were respectively used to construct cloned embryos. The results showed that the cleavage rate of recombinant embryos in BEF422 cells was significantly (P 0.05) higher than in EGFP-Nanog expressing cells (82.14 vs 40.38%), but the blastocyst development rate in the latter was slightly higher than in the former (17.30 vs 14.29%) (P 0.05), indicating that Nanog-overexpressed fibroblasts may be a better candidate of donor cells. To our knowledge, this is the first time that Nanog gene has been introduced into fibroblast cells to produce cloned embryos in bovine.
Keywords :
Bovine , Development competence , Eukaryotic expression , Molecular cloning , Nanog , Somatic nuclear transfer
