Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells transfected with enhanced green fluorescence protein gene.
We assessed the developmental ability of embryos cloned from porcine neural stem (NS) cells, amniotic fluid-derived stem (AFS) cells, fetal fibroblast cells, adult fibroblast, and mammary gland epithelial cells. The five cell lines were transfected with enhanced green fluorescence protein gene respectively using lipofection. NS and AFS cells were induced to differentiate in vitro. Stem cells and their differentiated cells were harvested for analysis of the markers using RT-PCR. The five cell lines were used for nuclear transfer. The two-cell stage-cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that both NS and AFS cells expressed POU5F1, THY1 and SOX2, and they were both induced to differentiate into astrocyte (GFAP+), oligodendrocyte (GalC+), neuron (NF+, ENO2+, and MAP2+), adipocyte (LPL+ and PPARG-D+), osteoblast (osteonectin+ and osteocalcin+), myocyte (MYF6+ and MYOD+), and endothelium (PECAM1+, CD34+, CDH5+, and NOS3+) respectively. Seven cloned fetuses (28 days and 32 days) derived from stem cells were obtained. The in vitro developmental ability (morula-blastocyst rate was 28.26-30.07%) and in vivo developmental ability (pregnancy rate were 1.67-2.17%) of the embryos cloned from stem cells were higher (P<0.05) than that of the embryos cloned from somatic cells (morula-blastocyst rate was 16.27-19.28% and pregnancy rate was 0.00%), which suggests that the undifferentiated state of the donor cells increases cloning efficiency.
Zheng
Yue-Mao
YM
College of Veterinary Medicine, Institute of Biotechnology, Northwest A&F University, 1-06#, Research Building, Yangling, Shaanxi 712100, People's Republic of China. zhengyuemao@163.com
Zhao
Hui-Ying
HY
Zhao
Xiao-E
XE
Quan
Fu-Sheng
FS
Hua
Song
S
He
Xiao-Ying
XY
Liu
Jun
J
He
Xiao-Ning
XN
Lin
Hui
H
D000653:Amniotic Fluid / Q000166:cytology*; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D019070:Cell Lineage; D019976:Cloning, Organism / Q000379:methods; D046149:Embryo Culture Techniques; D004624:Embryo Transfer; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008321:Mammary Glands, Animal / Q000166:cytology*; D058953:Neural Stem Cells / Q000378:metabolism*; D053652:Nuclear Transfer Techniques; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D018873:Pregnancy Rate; D013234:Stem Cells / Q000378:metabolism*; D013552:Swine; D014162:Transfection
D005819:Genetic Markers; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins