ABSTRACT
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.