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Design and Application of a shRNA 215simple transfection-based assay. Typically, it is found that 20–30% of sequenceverifiedshRNAs efficiently knockdown their target. Therefore, if 3–6 aretested, at least one should knockdown its target.To generate shRNAs, the U6-shRNA fusion is synthesized by PCR asdescribed in section 3.1.1.–3.1.7. and subcloned into pPUR V2 as an EcoRI/XhoIfragment. This plasmid is transfected into cells that express the target gene (typicallyU2OS osteosarcoma cells, because they are readily transfectable), the transfectedcells are selected for 48 h in 1 µg/mL puromycin and then targetknockdown is assayed by protein Western blot and/or reverse transcriptase (RT)-PCR of the mRNA. Plasmid pPUR V2 was designed for this purpose (Fig. 1). Inthis plasmid, selection of transfected cells with puromycin is rapid and the customdesignedMCS allows functional shRNAs to be easily shuttled from pPUR V2 intoa unique NheI (New England Biolabs, Ipswich, MA) site in the 3′-long terminalrepeat (LTR) of the retrovirus (pQCXIP, pQCXIH, or pQCXIN [Clontech]).1. Design and order PCR primers: the reverse primer encoding the shRNA isdesigned using “RNAi Central” at http://katahdin.cshl.org:9331/RNAi_web/scripts/main2.pl. Click on “shRNA design.” Using the radio buttons anddrop-down menu, select three “29-mer design sense–antisense” and three “29-merantisense– sense.” Make sure that the accession numbers or sequences that matchcDNA or exon sequences are entered. The website generates sequences of oligosencoding the shRNA and an EcoRI site for subcloning into pPUR V2. A GCGCsequence should be added to the 5′-end of the oligo to facilitate digestion byEcoRI. For each target gene, reverse primers containing the shRNA sequenceshould be synthesized at 0.05-µmol scale by Sigma-Genosys (The Woodlands,TX) or elsewhere. In addition, the forward PCR primer that is common to allshRNAs and contains a XhoI site and a SP6 primer should be synthesized: 5′-GGCCCTCGAGGATTTAGGTGACACTATAG-3′. Additional information onshRNA design is at RNAi Central.2. Perform PCR to generate the U6-shRNA cassette: this is schematized in Fig. 2. Asa PCR template, the pGEM1-U6 plasmid containing the human U6 RNA polymeraseIII promoter is used. Set up the PCR reaction, as follows:pGEM1-U61 ng50 mM MgCl 22.5 µL2.5 mM Deoxynucleotide 5′-triphosphate 4 µL10X Taq buffer 5 µL40% Dimethyl sulfoxide 5 µL50 µM SP6 1 µL50 µM Hairpin primer 1 µLddH 2O 30 µLTaq DNA polymerase 1 µLDeep vent DNA polymerase 0.2 µLFinal volume 50 µL