Amplification of BDNF and VEGF165 genes BDNF gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR, and the size of BDNF gene was 744 bp. The VEGF165 gene was obtained from pIRES2-VEGF165-EGFP plasmid by PCR, and the size of VEGF165 gene was 576 bp (Figure 1). Identification of plasmid pIRES2-BDNF-EGFP The plasmid pIRES2-BDNF-EGFP was cut by Eco RⅠand Bam HⅠdouble enzyme. A gene fragment with 744 bp was obtained, which was in full agreement with BDNF gene (Figure 2).  Identification of plasmid pIRES2-BDNF-VEGF165 The plasmid pIRES2-BDNF-VEGF165 was cut by Eco RⅠand Bam HⅠ double enzyme. And a fragment about 744 bp would be obtained. This indicated BDNF gene inserted into the plasmid pIRES2-BDNF-VEGF165. The plasmid pIRES2-BDNF-VEGF165 was cut by Bam HⅠand NotⅠdouble enzyme, and a fragment about      1 164 bp would be obtained, indicating IRES-VEGF165 gene could be inserted into the plasmid pIRES2-BDNF-VEGF165. The plasmid pIRES2-BDNF-VEGF165 was cut by Eco RⅠand NotI double enzyme, and a fragment about 1 914 bp would be obtained, indicating BDNF-IRES-VEGF165 gene inserted into the plasmid pIRES2-BDNF-VEGF165. The sequence of the plasmid pIRES2-BDNF-VEGF165 was in accordance with gene sequence in Gene Bank   (Figure 3)."

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To illustrate mRNA expression by pIRES2-EGFP, pIRES2-BDNF/EGFP and pIRES2-BDNF/VEGF165 transfected HEK293 cells, we evaluated the expression of BDNF and VEGF165 by RT-PCR analysis. RT-PCR was performed using BDNF-specific primers and the β-actin sequence served as an internal standard. GFP expression was monitored in pIRES2-EGFP and pIRES2-BDNF/EGFP transfected HEK293 cells by inverted fluorescence microscopy. Expression of BDNF mRNA was higher in either pIRES2-BDNF/EGFP or pIRES2-BDNF/VEGF165 transfected HEK293 cells than that pIRES2-EGFP transfected HEK293 cells or negative controls (P < 0.01). As shown above, expression of VEGF165 mRNA was higher in pIRES2-BDNF/VEGF165 transfected HEK293 cells than the other three (P < 0.01, Figure 5). These results demonstrated that the BDNF and VEGF165 had been introduced successfully into HEK293 cells by pIRES2-BDNF/EGFP and pIRES2-BDNF/VEGF165."

Western blot analysis of the expression of BDNF and VEGF165 in HEK293 cells   The results suggested that exogenous BDNF protein was strongly expressed in pIRES2-BDNF/EGFP and pIRES2-BDNF/VEGF165 transfected HEK293 cells, but the pIRES2-EGFP transfected HEK293 cells and non-transfected expression of endogenous BDNF was very low (Figure 6). As shown above, western blot analysis also revealed that exogenous VEGF165 protein was strongly expressed in pIRES2-BDNF/VEGF165 transfected HEK293 cells, but endogenous VEGF165 very low in the pIRES2-EGFP, pIRES2-BDNF/EGFP transfected HEK293 cells (Figure 7). These results also demonstrated that BDNF and VEGF165 had been introduced successfully into HEK293 cells by pIRES2-BDNF/VEGF165."