[Construction of adenoviral vector encoding human VEGF(121) cDNA and its expression in vitro].
OBJECTIVE
To construct the adenoviral vector bringing hVEGF(121) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.


METHODS
Human vascular endothelial growth factor (hVEGF(121)) cDNA obtained from the plasmid pCDI/VEGF(121) was cloned into plasmid pshuttle and further cloned to Adeno-X Viral DNA. The recombinant adenoviral plasmid was identified and then transferred to the adenoviral packaging cell HEK293 by lipofectamine mediated gene transfer method to pack the virus. After titilating the virus, the mouse bone marrow stromal cells (MSC) were transfected by the adenovirus and the expression of VEGF gene was detected.


RESULTS
The recombinant Adeno-VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. After MSCs were tranfected by the virus, RT-PCR showed that hVEGF(121) mRNA was transcripted from the hVEGF(121) gene. Western blot and immune histochemistry showed VEGF(121) protein was expressed in transgene MSCs.


CONCLUSIONS
The recombinant adenoviral vector bringing hVEGF(121) cDNA was successfully constructed and the transgene MSC expressed hVEGF gene in vitro, it provided the further foundation of VEGF gene therapy for bone ischemic diseases.
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
D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D018076:DNA, Complementary; D016228:Endothelial Growth Factors; D008222:Lymphokines; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors