In
Vitro
Development Competence of Bovine Nuclear Transfer Embryos Derived
from Nanog-Overexpressing Fibroblast Cells
Xi-bang Zheng§, Yan Yun, Yong-ce Hu, Yong Li1, Hua-yan Wang, Xiao-ling
Ma, Jin-qiang
Sui, An-min
Lei and Zhong-ying
Dou*
Shaanxi Branch of National Stem Cell Engineering
and Technology Center, Animal Medicine College, Northwest University
of Agriculture and Forestry, NO.3 Taicheng Road,Yangling712100,
Shaanxi, P.R.China; 1College
of Life Science, Ningxia University, NO.489 Helanshan
West Road, Yinchuan 750021, Ningxia, P.R. China §Also
affiliated with Department of Animal Medicine, College of Animal
Science and Technology, Guangxi University, NO.100 Daxue Road, Nanning 530005,Guangxi, P.R. China *Corresponding author:
douzhongying@china.com
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.
Key words:
Bovine, Development competence,
Eukaryotic expression, Molecular cloning,
Nanog, Somatic nuclear transfer