[Expression of VEGF165 and VEGF121 genes in MC3T3-E1 cell line and biological function of their products].
To construct vectors stably expressing bioactive VEGF165 and VEGF121 in MC3T3-E1 cells.



VEGF165 and VEGF121 genes were obtained by RT-PCR from HL-60 cells and inserted into eukaryotic expression plasmid pcDNA3.1(+). The constructed plasmids containing right sequence of target genes were transfected into MC3T3-E1 cells by lipofectin. The cells were limitedly diluted twice and selected by G418, the expression of VEGF was tested by ELISA, Western-blot and immunofluorescence essay. The bioactivity of expressed products was proved by MTT method.



DNA sequencing indicated the sequences of the obtained VEGF165 and VEGF121 were identical to the those reported in Genebank, and the newly-constructed plasmids were VEGF165/pcDNA3.1(+) and VEGF121/pcDNA3.1(+). The results of ELISA, Western-blot and immunofluorescence showed that the transfected cells could express VEGF165 and VEGF121 proteins. And MTT proved the proteins were bioactive.



The transfected MC3T3-E1 cells could stably express high levels of bioactive VEGF165 and VEGF121.
He
Jun
J
The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Wang
Hui-ming
HM
Yao
Hang-ping
HP
D016475:3T3 Cells; D000818:Animals; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A