Abstract:

BACKGROUND: Alpha-smooth muscle actin gene is one of several genes expressed in smooth muscle cells and has been recognized as the marker for smooth muscle cell phenotype transformation.
OBJECTIVE: To construct a recombinant pLVpuro/αSMA-hrGFP lentiviral vector by multisite Gateway technology.
METHODS: Primers containing attB sites were designed and used to amplify the alpha-smooth muscle actin (αSMA) promoter fragment by PCR from the plasmid containing the mouse αSMA promoter sequence (SMP8-Cre). By the BP recombination reaction, the attB flanked PCR product containing αSMA promoter sequence was cloned to an attP-containing pDONR P4P1r donor vector to create an entry clone, pUp-αSMA. Finally, pUp-αSMA and pDown-hrGFP were shuttled into the destination vector pDEST-puromycin by LR recombination reaction to generate pLVpuro/αSMA-hrGFP. The expression vector was confirmed by PCR and gene sequencing. Then this expression vector was transferred into the C2C12 cell line, and the gene expression was confirmed by immunofluorescent staining.
RESULTS AND CONCLUSION: The pLVpuro/αSMA-hrGFP expression vector was successfully constructed with the right αSMA promoter fragment confirmed by sequencing. The activity of αSMA promoter was verified by cell transfection and immunofluorescent staining. The recombinant pLVpuro/αSMA-hrGFP lentivector was successfully constructed. And it offers an important tool to monitor the differentiation process from embryonic stem cells to vascular smooth muscle cells, and for cell tracing and gene function studies.