Adenoviral Vector Construction for GLI1 Expression

Smoothened agonist (SAG) was purchased from Calbiochem (San Diego, CA; 566660). Receptor activator of NF-kappa-B ligand (RANKL) was purchased from Pepro Tech (Rocky Hill, NJ; 184-01791). Sonic hedgehog (Shh) was purchased from R&D Systems (Minneapolis, MN; 1845-SH). Cyclopamine was purchased from Enzo Life Sciences (Farmingdale, NY; BML-GR3334). Plasmids expressing human GLI1 were constructed as previously described [12] (link). The adenoviral vector expressing human GLI1-Biotin-3xFLAG-IRES-dsRed was constructed using the pAd/PL-DEST vector and ViraPower Adnoviral Expression System (Life Technologies, Carlsbad, CA), according to the manufacturer's instructions. In brief, human GLI1 cDNA carrying Biotin-3xFLAG tag [16] (link) was initially cloned into pCID vectors [17] (link); GLI1-Biotin-3xFLAG-IRES-dsRed was then transferred into the pENTR1A vector in conjunction with the CAGGS promoter and subjected to adenoviral vector construction using the Gateway system. The pAd/PL-DEST expressing the CAGGS promoter-driven GLI1-Biotin-3xFLAG-IRES-dsRed was linearized with Pac I and transfected into 293A cells. After amplification, the virus was stored at −80°C. The viral titer was determined by an end-point titer assay using 293A cells.