Figure 1. Construction of the pLVTHM vector encoding anti-GLI1 shRNA.

For the GLI1-shRNA vector, two single strand DNAs encoding two linkers, the target sequences and a loop element, were synthesized. These were annealed to double stranded DNA, and ligated into the pLVTHM following MluI and CalI digestion. The short hairpin form of shGLI1 is expressed under the control of human H1 promoter. The vector also contains a human EF1-α promoter driving the GFP marker gene for tracking transduced cells. The vectors were generated by transient transfection of pRsv-REV, pMDlg-pRRE, pMD2G, and the shRNA encoding pLVTHM into 293T cells. Abbreviations: RRE, Rev response element; cPPT, central polypurine tract; EF1-α, human elongation factor 1-α promoter; H1, the human H1 promoter; GFP, green fluorescent protein; PRE, human hepatitis virus posttranscriptional regulatory element.