[Construction of lentiviral vector pTK208-mVEGF-IRES-DsRed contains dsred and mVEGF and its expression in 293T cells].
OBJECTIVE
To construct red fluorescent protein (DsRed) and mouse vascular endothelial growth factor (mVEGF) lentiviral expression vector in order to obtain the DsRed and VEGF expression in 293T cells.


METHODS
The mVEGF was amplified from the lungs of fetal mouse by RT-PCR. The IRES-DsRed was obtained from plasmid pLV-tTRKRAB-red which contains IRES-DsRed sequence by PCR. Then they were cloned into lentiviral expression vector pTK-208. Human embryonic kidney 293T cells were co-transfected with the three plasmids by lipofectamine, and the expression of DsRed was examined under fluorescent microscope 24 h and 48 h after transfection. 48 h and 72 h later the viral supernatant was used to infect 293T cells, the expression of DsRed was examined under fluorescent microscope and Western blot assays was used to examine the expression of mVEGF in cells and the culture medium.


RESULTS
The recombinant lentiviral vector pTK208-mVEGF-IRES-DsRed was constructed, the virus titres were above 10(6) PFU/mL in the supernatant. After infection, DsRed and mVEGF proteins were successfully expressed in 293T cells.


CONCLUSIONS
The lentiviral vector pTK208-mVEGF-IRES-DsRed contains DsRed and mVEGF were successfully constructed and expressed in 293T cells, it could provide a basis for further researches on the pathophysiological mechanism of VEGF and lay the foundation of gene therapy.
Wang
Dong-yang
DY
Laboratory of Transplant Immunology, Xuzhou Medical College, Xuzhou 221002, China.
Jia
Lu
L
Chen
Chong
C
Li
Yan-jie
YJ
Zeng
Ling-yu
LY
D000818:Animals; D004622:Embryo, Mammalian; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D000097573:Red Fluorescent Protein; C467480:vascular endothelial growth factor A, mouse