Abstract:

BACKGROUND: Construction of a lentiviral vector concurrently containing a specific gene, a fluorescent reporter and an antibiotic selection marker under the control of different constitutive promoters for gene overexpression studies has not been reported before.
OBJECTIVE: To construct the lentivector, pLVpuro/EF1α-PEDF-IRES-EGFP, which containing the elements of PEDF and EGFP gene driven by EF1α promoter and puromycin resistant gene driven by PGK promoter through multisite gateway technology and to transduce it into human mesenchymal stem cells (hMSCs) to detect the protein expression of PEDF gene.
METHODS: The PEDF cDNA was flanked with attB sites by PCR and then cloned into pDONRTM221 to generate pDown-PEDF by utilizing the BP recombination reaction. The entry clones, pUp-EF1α, pDown-PEDF, pTail-EGFP were then recombined into the destination vector pDEST-puromycin to construct expression lentiviral vector, pLVpuro/EF1α-PEDF-IRES-EGFP by LR recombination reaction. The lentiviral particles were prepared by transient co-transfection of the resulting vector and ViraPower™ Lentiviral Packaging Mix into 293FT cells using Lipofectamine 2000. The viral particles were harvested concentrated and used to infect hMSCs. 7 days after transduction, 1-5 mg/L puromycin was added to the culture and lasted for 5 days. Transduced hMSCs were purified and then analyzed by Western Blot and ELISA assay.
RESULTS AND CONCLUSION: In this study, the lentivector pLVpuro/EF1α-PEDF- IRES-EGFP was successfully constructed by multisite gateway technology and it was confirmed by PCR and gene sequencing and the hMSCs were successfully transduced by lentivirus. After antibiotic selection, pure infected cell population which expressed PEDF gene and EGFP were attained.