shRNA gene silencing Rat - MM1 ADF

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi has been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining the efficacy of transduction and shRNA on its target site.

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The plasmids for retroviral expression were constructed by subcloning the cDNAs encoding cyan fluorescent protein (CFP), or CFP-tagged human SSH1 (CFP-hSSH1), mouse SSH2 (mSSH2-CFP), human wild-type (WT) LIMK1 (hLIMK1(WT)-CFP), or human kinase-dead LIMK1 (hLIMK1(D460A)-CFP) (30), or Myc-tagged chick cofilin (ch-cofilin) or ADF (ch-ADF), into the pLNCX-neo retroviral vector (Clontech, Palo Alto, CA). The siRNA-targeting constructs were generated using the pSUPER.retro.puro vector (Oligo-Engine, Seattle, WA), as described previously (32). The 19-base targeting sequences used in this study were as follows: 5′-GAACTGTCGACCAAGAAAG-3′ (rat SSH1), 5′-TGCGTCAAACTTAGAGGAC-3′ (rat SSH2), 5′-GCTGGAACAATGGCTAGAA-3′ (rat LIMK1), 5′-GCACGAATTACAAGCTAAC-3′ (rat cofilin), and 5′-GCACGAGTATCAAGCAAAT-3′ (rat ADF). As a control, we used a nontargeting sequence, 5′-TCTTCCCCCAAGAAAGATA-3′, which does not exist in the rat genome.To generate retroviral supernatants, Plat-E retrovirus packaging cells (33) were transfected with pLNCX-neo or pSUPER.retro.puro plasmids using FuGENE6 (Roche Applied Science, Mannheim, Germany). At 48 h after transfection, the culture medium was centrifuged, and the viral supernatant was used for infection after the addition of 8 μg/ml polybrene. Infected MM1 cells were cultured for 24 h, washed, and selected by culturing for 48 h with 2 μg/ml puromycin for cells infected with pSUPER.retro.puro retrovirus or for 2 weeks with 250 μg/ml G418 (Nacalai Tesque, Kyoto, Japan) for cells infected with pLNCX retrovirus.
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