Construction of VEGF₁₆₅ lentivirus vectors and expression in adipose tissue-derived stem cells after transfection

OBJECTIVE: To clone target gene by RT-PCR method, construct VEGF₁₆₅ lentivirus vectors, transfect adipose tissue derived stem cells (ADSCs) and verify the expression of VEGF₁₆₅ in vitro and in vivo. METHODS: RT-PCR technology was employed to clone VEGF₁₆₅ gene, and this gene was cloned to lentivirus vector pLVX-EF1α-IRES2-AcGFP1 to construct a lentiviral vector pLVX-EF1α-VEGF₁₆₅-IRES2-AcGFP1. Bacterial colonies PCR and sequencing analysis were used for identification. Then, Lenti-X 293T cells were transfected with main vector pLVX-EF1α-VEGF₁₆₅-IRES2-AcGFP1, packaging plasmid gag-pro, vpr-pol, Tet-Off^™, tat-IRES-rev and coating plasmid env (VSV-G). Lentiviral vectors were packaged and the titer was determined. ADSCs were isolated by collagenase digestion method, then cultured, and identificated by morphology, immunofluorescence and multi-directional differentiation. ADSCs was transfected with packaged VEGF₁₆₅ lentivirus. Immunofluorescence and ELISA were used to detect the expression of VEGF₁₆₅ in vitro. ADSCs transfected with VEGF₁₆₅ lentivirus were injected into nude mice. ELISA was used to detect the expression of VEGF₁₆₅. RESULTS: The VEGF₁₆₅ gene fragment was cloned successfully, and the lentiviral vector plasmid pLVX-EF1α-HVEGF₁₆₅-IRES2-AcGFP1 was confirmed to contain VEGF₁₆₅ gene fragment as shown by bacterial colonies PCR. DNA sequencing analysis confirmed that VEGF₁₆₅ gene sequencing was exactly the same with that reported by Genbank. After transfection, a large number of Lenti-X 293T cells with green fluorescence were observed by fluorescent microscopy. The concentration of the virus titer was 1×10⁸·TU·mL^-1. ADSCs were identified by morphology, immunofluorescence and multi-directional differentiation methods, in line with the literature reported ADSCs characteristics. There were about 90% of ADSCs which could express VEGF₁₆₅ after being transfected with the viruses by immunofluorescence detection, also, VEGF₁₆₅ protein was proved by ELISA to express stably and efficiently in vitro and in vivo. CONCLUSION: Lentiviral vectors expressing VEGF₁₆₅ are successfully constructed by cloning target gene with RT-PCR method. After transfection, ADSCs expressing VEGF165 protein stably in vitro and in vivo can be obtained.