Construction and identification of pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector

doi:10.3969/j.issn.2095-4344.2014.29.015

Abstract

Abstract:

BACKGROUND: Human glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor 165 (VEGF₁₆₅) are essential genes for cell differentiation.

OBJECTIVE: To construct and identify pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector.

METHODS: Human GDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then the GDNF cDNA fragment was inserted into the multiple cloning sites of pIRES₂-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES₂-GDNF-EGFP. The VEGF₁₆₅gene was obtained from pIRES₂-VEGF₁₆₅-EGFP plasmid by twin PCR. Then VEGF₁₆₅cDNA fragment was cloned into the pIRES₂-GDNF-EGFP, instead of EGFP, to create a double gene co-expressing vector plasmid pIRES₂-GDNF-VEGF₁₆₅ containing internal ribosome entry sites. Then pIRES₂-GDNF-VEGF₁₆₅was used to transfect HEK293 cells. RT-PCR and western blot analysis were performed to test the co-expression of double genes.

RESULTS AND CONCLUSION: DNA sequencing analysis demonstrated that the GDNF and VEGF₁₆₅ were exactly consistent with the sequence recorded in the GenBank. The size of GDNF gene was 636 bp and the size of VEGF₁₆₅ gene was 576 bp. Enzyme digestion analysis indicated that, pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector inserted GDNF band by Bgl II/Bam HI, inserted IRES-VEGF₁₆₅ fragment by Bam HI/Not I, and inserted GDNF-IRES-VEGF₁₆₅ fragment by Bgl II/Not I. RT-PCR and western blot analysis showed that, after HEK293 cells were transfected with pIRES₂-GDNF-VEGF₁₆₅, double genes were expressed at the mRNA and protein levels. The pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector is successfully constructed.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: glial cell line-derived neurotrophic factor, vascular endothelial growth factor, carrier proteins, tissue engineering

CLC Number:

R318

Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen. Construction and identification of pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(29): 4675-4682.

Figures/Tables 2

Amplification of GDNF and VEGF165 genes GDNF gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by overlap PCR. The size of GDNF gene was 636 bp. The VEGF165 gene was obtained from pIRES2-VEGF165-EGFP plasmid by twin PCR, and the size of VEGF165 gene was 576 bp (Figure 1). Identification of plasmid pIRES2-GDNF-EGFP The plasmid pIRES2-GDNF-EGFP was cut by Bgl II and Bam HI double enzyme. A gene fragment with 636 bp was obtained, which was in full agreed with GDNF gene (Figure 2A). Identification of plasmid pIRES2-GDNF-VEGF165 The plasmid pIRES2-GDNF-VEGF165 was cut by Bgl II and Bam HI double enzyme. And a fragment about 636 bp would be obtained. This indicated GDNF gene inserted into the plasmid pIRES2-GDNF-VEGF165. The sequence of the plasmid pIRES2-GDNF-VEGF165 was in accordance with gene sequence in Gene Bank. The plasmid pIRES2-GDNF-VEGF165 was cut by Bam HI and Not I double enzyme. And a fragment about 1 183 bp would be obtained. This indicated IRES-VEGF165 gene inserted into the plasmid pIRES2-GDNF-VEGF165. The plasmid pIRES2-GDNF- VEGF165 was cut by Bgl II and Not I double enzyme. And a fragment about 1 825 bp would be obtained. This indicated BDNF-IRES-VEGF165 gene inserted into the plasmid pIRES2-GDNF-VEGF165. The sequence of the plasmid pIRES2-BDNF-VEGF165 was in accordance with gene sequence in Gene Bank (Figure 2B). RT-PCR analysis of the expression of GDNF and VEGF165 in HEK293 cells To illustrate mRNA expression by pIRES2-EGFP, pIRES2-GDNF/EGFP, and pIRES2-GDNF/VEGF165 transduced HEK293 cells, we evaluated the expression of GDNF and VEGF165 by RT-PCR analysis. RT-PCR was performed using GDNF-specific primers and the β-actin sequence as an internal standard. GFP expression was monitored in pIRES2-EGFP and pIRES2-GDNF/EGFP transduced HEK293 cells by inverted fluorescence microscopy. Expression of GDNF mRNA was higher in either pIRES2-GDNF/EGFP or pIRES2-GDNF/VEGF165 transduced HEK293 cells than that pIRES2-EGFP transduced HEK293 cells or negative control (Figure 3A, B). As shown above, expression of VEGF165 mRNA was higher in pIRES2-GDNF/VEGF165-transduced HEK293 cells than the other three (Figure 3C, D). These results demonstrated that the GDNF and VEGF165 had been introduced successfully into HEK293 cells by pIRES2-GDNF/EGFP and pIRES2-GDNF/VEGF165. After the HEK293 cells were transduced by pIRES2-GDNF/EGFP, we passaged them continually and then monitored the mean percentage of expression of GFP under fluorescence microscopy. There was no decrease in GFP fluorescence, illustrating the maintenance of transgenic expression in the transduced cells. Western blot analysis of the expression of GDNF and VEGF165 in HEK293 cells To illustrate protein expression by pIRES2-EGFP, pIRES2-GDNF/EGFP, and pIRES2-GDNF/VEGF165 transduced HEK293 cells, we evaluated the expression of GDNF and VEGF165 by western blot assay. After 72 hours of transfection, we collected the culture supernatants of infected HEK293 cells without serums which were processed for western blot analysis using an anti-GDNF and anti-VEGF165 antibody. β-actin served as an internal standard. The results suggested that exogenous GDNF protein was strongly expressed in pIRES2-GDNF/EGFP and pIRES2-GDNF/VEGF165 transduced HEK293 cells, but the pIRES2-EGFP transduced HEK293 cells and no transduced cells, the expression of endogenous GDNF was very low (Figures 4A, B). As shown above, western blot analysis also revealed that exogenous VEGF165 protein was strongly expressed in pIRES2-GDNF/VEGF165 transduced HEK293 cells, and expression of endogenous VEGF165 was very low in the pIRES2-EGFP, pIRES2-GDNF/EGFP transduced HEK293 cells (Figures 4C, D). These results also demonstrated that GDNF and VEGF165 had been introduced successfully into HEK293 cells by pIRES2-GDNF/VEGF165. "

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Construction and identification of pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector

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