[Construction of human growth hormone lentiviral vector and its expression in murine skeletal myoblasts].
The aim of this study is to construct a lentiviral vector encoding human growth hormone, and to achieve the long, efficient and stable expression in murine skeletal myoblasts. Primary skeletal myoblasts were isolated from Sprague-Dawley rats and cultured by enzymatic digestion. We tested them by Desmin immunohistochemistry stains and found their viability was up to 94% by Trypan blue. Human growth hormone (hGH) cDNA was subcloned into expression vector pLenti6/V5-D-TOPO to construct recombinant pLenti6/V5-hGH. The pLenti6/V5-hGH and the contructed pLenti6/V5-EGFP were transfected into murine skeletal myoblasts by the Lipofectamin 2000. Through counting by the Confocal Laser Scanning Microscope, we identified the transfection efficency. We added the blasticidin to the 6-well plate with lids and obtained stable myoblasts expressing hGH. The concentration of human growth hormone (hGH) in cell culture medium was detected by Radioimmunoassay (RIA). Polymerase Chain Reaction (PCR) and DNA sequence showed hGH cDNA had been correctly inserted into pLenti6/V5-D-TOPO vector. Bright green fluorescence of the transfected cells could be observed under the Confocal Laser Scanning Microscope after 24 h transfection with pLenti6/V5-EGFP plasmids, and the transfection rate reached 40%. The difference was distinct (P < 0.01) between the pLenti6/V5- hGH groups and control groups in the secretive level of human growth hormone. After 8 weeks, the expression of human growth hormone was still stable. Then, we validated the biological characterization of the rhGH by the enzyme-link immunosorbent assay (ELISA) of the Insulin-like growth factor I (IGF-1). These results demonstrate we have successfully constructed the recombinant pLenti6/V5-hGH plasmids and accomplished rhGH long, efficient and stable expression ectopic in skeletal muscle myoblasts.
Liu
Xiang-Yang
XY
Department of cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. longliu0714@163.com
Lu
Yong-Xin
YX
Xu
Yu-Lan
YL
Li
Xiao-Qing
XQ
Liu
Juan
J
Li
Ai-Hua
AH
Luo
Ping
P
Wan
Jian-Ping
JP
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D005822:Genetic Vectors; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism*; D032448:Myoblasts, Skeletal / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; D019382:Human Growth Hormone