Adenovirus-Based Short Hairpin RNA Vectors Containing an EGFP Marker and Mouse U6, Human H1, or Human U6 Promoter (2005)

BioTechniquesVol. 38, No. 4 RNA TechnologiesOpen AccessAdenovirus-based short hairpin RNA vectors containing an EGFP marker and mouse U6, human H1, or human U6 promoterSeungil Ro, Sung Jin Hwang, Tamás Ördög & Kenton M. SandersSeungil Ro*Address correspondence to: Seungil Ro, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA. e-mail: E-mail Address: ro@unr.eduUniversity of Nevada School of Medicine, Reno, NV, USA, Sung Jin HwangUniversity of Nevada School of Medicine, Reno, NV, USA, Tamás ÖrdögUniversity of Nevada School of Medicine, Reno, NV, USA & Kenton M. SandersUniversity of Nevada School of Medicine, Reno, NV, USAPublished Online:30 May 2018https://doi.org/10.2144/05384RN01AboutSectionsView ArticleSupplemental MaterialPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail View article 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 colony-direct PCR. The products can be distinguished by size: either 424 bp for pAd shRNA/H1 with a shRNA duplex insert or 355 bp without it. The primers can also be used for direct sequencing of the PCR products. These vectors also contain the EGFP cassette controlled under the CMV promoter, which allows monitoring of transfection or transduction at the single cell level.Figure 1. Construction of the adenovirus-based short hairpin RNA (shRNA) vectors.Three adenoviral shRNA vectors (pAd shRNA/mU6, pAd shRNA/hU6, and pAd shRNA/H1), each containing an shRNA cassette under the control a different RNA polymerase III (RNA pol III) promoter (mU6, hU6, or H1), were constructed. The vector expresses enhanced green fluorescent protein (EGFP) as a marker for transfection or transduction. An insert of 378 bp can be digested out by BamHI and HindIII and replaced with an shRNA oligonucleotide duplex (shown in the box). If N1 [the +1 position of the target small interfering RNA (siRNA)] is C or T, a G must be added before N1. RITR, left Ad5 inverted terminal repeat; Ori, pBR322 origin of replication; Kan, kanamycin resistance open reading frame (ORF); LITR, right Ad5 inverted terminal repeat; ES, encapsidation signal; Pol III, RNA pol III U6 or H1 promoter; CMV, cytomegalovirus promoter; Poly A, simian virus 40 (SV40) poly(A) signal; Right arm, Ad5 right arm homology; Left arm, Ad5 left arm homology.We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing in mouse jejunal cells as a model to test the efficiency of these vectors. The GAPDH shRNA control insert obtained from Ambion (Austin, TX, USA) is annealed to double-stranded oligonucleotides with BamHI and HindIII sticky ends surrounding the shRNA template that targets the GAPDH messenger RNA (mRNA) (see the supplementary materials). This annealed insert was ligated into the three vectors. A negative control nontargeting shRNA was also inserted into the hU6 vector. Adenoviruses were produced from these constructs using the AdEasy™ Adenoviral Vector System (Stratagene), according to the manufacturer's instructions (www.stratagene.com/manuals/240009.pdf). The titer was 1-5×1011 plaque-forming unit (pfu)/mL.The adenoviruses were used to transduce primary cultures of mouse jejunal tunica muscularis cells. The cells were transduced with adenoviruses expressing either negative control shRNA or GAPDH shRNA. Two days after transduction, fibroblast cells and smooth muscle cells were all GFP positive, but network-forming cells showed about 10%-20% of cells GFP positive (data not shown). Some cell types such as skeletal muscle cells, endothelial cells, hematopoietic cells, and many tumor cells are known to be resistant to adenovirus infection due to a lack of the coxsackievirus and adenovirus receptor (CAR) (3). Several researchers have shown adenovirus transduction improved with the aid of cationic lipids, polymers, liposomes, EGTA, sodium caprate, or calcium phosphate (4–10). However, these reagents are toxic to primary cells and kill most of them after transduction. To improve transduction, we used an advanced nonliposomal transfection reagent, FuGENE™ 6 (Roche Applied Science, Indianapolis, IN, USA), in the amount of 3 µL FuGENE 6 plus 109 pfu viruses in a 6-well plate. This method showed minimal toxicity along with a high efficiency of transduction. Using this method, more than 90% of network-forming cells, as well as other cell types transduced with the negative control adenovirus and FuGENE 6, were GFP positive (Figure 2). The same results were observed with cells transduced with the GAPDH shRNA-expressing adenovirus.Figure 2. Enhanced green fluorescent protein (EGFP) monitoring of the transduction efficiency of recombinant adenoviral vector in cultured mouse jejunal tunica muscularis cells.Data are shown for cells transduced with the negative control short hairpin RNA (shRNA)-EGFP construct. (A) Network-forming cells in the primary cultures visualized by near-infrared differential interference contrast (NIR-DIC). (B) EGFP fluorescence in the same field-of-view. (C) Composite image. Note strong expression of EGFP in network-forming cells in the primary culture. Scale bar in panel C applies to all panels.The cells transduced with the GAPDH shRNA-expressing adenovirus were harvested 2 days after transduction for protein analysis. GAPDH protein with a molecular mass of 37 kDa was detected by Western blot analysis. A representative blot is shown in Figure 3. The reduction levels of protein were 80% (±6%), 88% (±4%) and 75% (±8%) in the cells transduced with GAPDH shRNA/mU6, GAPDH shRNA/H1, and GAPDH shRNA/hU6, respectively, when compared to levels in the negative control shRNA-transduced cells (n=2). Thus, the H1 promoter was slightly more effective than either the mU6 or hU6 promoter for the mouse jejunal cells. β-Actin protein with a molecular mass of 45 kDa was used as an endogenous control. The levels of this protein did not change following treatment with adenovirus. The promoter activity of these three adenoviruses was also tested in NIH3T3 cells. As expected, the mU6 promoter was more efficient than the H1 or the hU6 promoter. Reductions in the GAPDH mRNA were 76% (±3%) with mU6, 65% (±4%) with H1, and 52% (±6%) with hU6, respectively (n=3).Figure 3. Short hairpin RNA (shRNA)-mediated reduction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in jejunal tunica muscularis cells.Lane 1, protein standard marker (Precision Plus Protein Standards; Bio-Rad Laboratories, Hercules, CA, USA); lane 2 (shNeg/hU6), negative control shRNA/hU6; lane 3 (shGAPDH/mU6), GAPDH shRNA/mU6; lane 4 (shGAPDH/H1), GAPDH shRNA/H1; lane 5 (shGAPDH/hU6), GAPDH shRNA/hU6. GAPDH protein is shown along with a molecular mass of 37 kDa (top). β-Actin protein was used as an endogenous control (bottom). The blot shows reduction of GAPDH protein levels in cells transduced with the GAPDH shRNA adenoviruses when compared to levels in negative control shRNA-transduced cells.In summary, we have constructed three new adenoviral shuttle vectors, pAd shRNA/mU6, pAd shRNA/H1, and pAd shRNA/hU6. This set of vectors has several advantages over other shRNA vectors. First, each vector contains an shRNA cassette that is under the control of a different promoter (mouse U6, the human H1, or U6 promoter), allowing users to select the most efficient shRNA system. Since all three constructs contain the same sites for cloning of the shRNA, the shRNA duplexes are easily interchangeable in all three vectors. Second, the use of screening primers allows us to pick up positive clones by a fast and accurate PCR-based technique. Third, the use of EGFP in the vector allows us to track the efficiency of transfection/transduction in mammalian cells.AcknowledgementsWe thank Drs. Kathleen D. Keef, Fiona Britton, and Wei Yan for insightful and critical comments on this manuscript and Rebecca Partain for technical support. This research has been supported by National Institutes of Health grant no. DK041315.Competing Interests StatementThe authors declare no competing interests.References1. Zhao, L.J., H. Jian, and H.H. Zhu. 2003. 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Kathleen D. Keef, Fiona Britton, and Wei Yan for insightful and critical comments on this manuscript and Rebecca Partain for technical support. This research has been supported by National Institutes of Health grant no. DK041315.Competing Interests StatementThe authors declare no competing interests.PDF download