Adenovirus-based short hairpin RNA vectors containing an EGFP marker and mouse U6, human H1, or human U6 promoter.

BioTechniques 625 Adenoviral shuttle vectors to express short hairpin RNAs (shRNA) using RNA polymerase III (RNA pol III) promoters (mouse U6, human H1, or human U6) have been constructed in several laboratories including Welgen (Worcester, MA, USA), BD Biosciences (San Jose, CA, USA), Invitrogen (Carlsbad, CA, USA), and GeneScript (Piscataway, NJ, USA). Welgen and Invitrogen developed adenovirus shuttle vectors to express shRNA under the control of the human U6 or H1 promoter. BD Biosciences developed a shuttle vector to express shRNA under the control of the human U6 promoter with red fluorescent protein (RFP) expression to track transfection efficiency. GenScript and Zhao et al. (1) also developed a shuttle vector under the control of the human H1 promoter with a green fluorescent protein (GFP) marker. The activities of RNA pol III-driven promoters vary with cell type (2). Therefore, the activities of these three different promoters should be tested to select the most efficient shRNA system. However, the cloning sites within these vectors among the different companies are different, and therefore, the shRNA oligonucleotide duplexes are not interchangeable. We constructed three new adenoviral vectors based on the shuttle plasmid pShuttle (Stratagene, La Jolla, CA, USA). The vectors (pAd shRNA/mU6, pAd shRNA/H1, and pAd shRNA/ U6) all contain an shRNA expression cassette and an enhanced GFP (EGFP) marker, and all three constructs have the same cloning sites (Figure 1). The shRNA cassette is controlled under the mouse U6, the human H1, or the human U6 RNA pol III promoter. In our constructs, a 378-bp placeholder fragment was inserted between the cloning sites (BamHI and HindIII) used for insertion of the shRNA. This produces a double digested product that is significantly different in size from the single digested product, making it easy to distinguish during gel elution. Another difficulty with cloning an annealed shRNA duplex into a vector is screening inserted positive clones. In our vectors, a pair of screening primers (P1 and P1r) is located upstream of the H1 or U6 promoters and at the cytomegalovirus (CMV) promoter for Adenovirus-based short hairpin RNA vectors containing an EGFP marker and mouse U6, human H1, or human U6 promoter