Construction and Identification of Lentiviral Vector Over-expressing Mouse miRNA-203

Objective To construct a lentiviral vector for the over-expression of microRNA-203(miR-203),and to explore its virus packaging,identification and titer.Methods miR-203 shRNA by chemical synthesis was inserted into pSicoR vector,and then identified by double digestion and nucleotide sequencing.Plasmids pSicoR-miR-203,pCMV-VSV-G and pCMV-dR8.91 were co-transfected into HEK-293T cells for packaging of the lentivirus using liposome,respectively,harvesting the supernatant in 48 hours and 72 hours.HEK-293T cells were then transduced with an appropriately diluted lentivirus supernatant for the titration of virus titer,and preparation of virus particles were injected Balb / c mice to detect miR-203 expression in vivo.Results Results from the DNA Digestion and sequencing confirmed that the recombinant plasmid pSicoR-miR-203 was successfully constructed.The titer of supernatant was 5×107 TU/mL.Recombinant virus was expressed at liver,spleen,lung and kidney in Balb/c mouse.Conclusion MiR-203 expression lentiviral vector was successfully constructed.It has laid an experimental basis on the miR-203 target validation and functional study.