Expression of human VEGF(121) cDNA in mouse bone marrow stromal cells.
OBJECTIVE
To construct a retroviral vector carrying human vascular endothelial growth factor (hVEGF (121)) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.


METHODS
hVEGF(121) cDNA was obtained from the plasmid pCDI/VEGF(121) and cloned into retroviral plasmid pLXSN. Recombinant plasmid was transferred to the retro virus packaging cell, PT-67, by lipofectamine mediated gene transfer. Mouse bone marrow stromal cells (MSCs) were transfected by the retrovirus. The integration of the hVEGF(121) cDNA into MSC genomic DNA and expression of the VEGF gene was detected. Proliferation assays of human umbilical vein endothelial cells (HUVECs) by VEGF(121) in culture medium were performed.


RESULTS
Recombinant pLXSN/VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. hVEGF(121) gene was integrated into MSC genomic DNA after transfection, and the VEGF(121) protein was expressed. Proliferation assays showed VEGF(121) in culture medium was a biologically active protein and had a mitogenic effect on HUVEC.


CONCLUSIONS
Recombinant retroviral vector carrying hVEGF(121) cDNA was successfully constructed. VEGF (121) protein expressed by MSCs had mitogenic effect biologically. This provides a further foundation for VEGF gene therapy for bone ischemic disease and bone tissue engineering.
Department of Orthopidics, Third Hospital, Peking University, Beijing 100083, China.
Shanbao
S
Cai
Qingjun
Q
Ma
Xijie
X
Yu
Gengting
G
Dang
Dalong
D
Ma
D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D002455:Cell Division; D018076:DNA, Complementary / Q000235:genetics*; D016228:Endothelial Growth Factors / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology; D015316:Genetic Therapy; D006801:Humans; D008222:Lymphokines / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D012190:Retroviridae / Q000235:genetics; D017154:Stromal Cells / Q000378:metabolism; D019076:Transgenes; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D019065:Virus Assembly
D018076:DNA, Complementary; D016228:Endothelial Growth Factors; D008222:Lymphokines; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors