Immortalization of bovine germ line stem cells by c-myc and hTERT.
The limited life span of bovine germ line stem cells in vitro is one of the obstacles to spermatogenesis analysis, genetic manipulation and generating transgenic animal. The aim of this study is to establish immortalized bovine germ line stem cells by c-myc or hTERT. We constructed pEMY and pETE expression vectors and transfected germ line cells from 5-month-old bovine. After G418 screening, four types of positive clones were obtained. The results showed that they expressed exogenous genes c-myc or hTERT at mRNA and protein level by RT-PCR and Western blotting detection. Presumable cell lines GM7, GT3, GMT5 all expressed germ-line-stem-cell-specific makers by immunocytochemical analysis, such as c-kit, Oct-4 and GFRalpha-1. The putative cell lines also had higher capacity of proliferation than freshly isolated bovine spermatogonial stem cells. So we can conclude that exogenous genes c-myc or hTERT have integrated into the genome of bovine germ cells and upregulated the expression of telomerase.
Bi
Cong-Ming
CM
Institute of Bio-engineering, Northwest Sci-Tech University of Agriculture and Forestry, No. 3 TaiCheng Road, Yangling 712100, PR China.
Zhang
Shi-Qiang
SQ
Zhang
Yong
Y
Peng
Shu-Ying
SY
Wang
Li
L
An
Zhi-Xing
ZX
Qi
Aladaer
A
Lv
Ning
N
D000818:Animals; D002417:Cattle / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism*; D007150:Immunohistochemistry; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors
D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; C509186:TERT protein, human; D019098:Telomerase