shRNA gene silencing Mouse - P19 Foxm1

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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4 years ago

4 years ago by A.C.Burton United Kingdom

Multiple gene silencing using ShRNA

Hello, can someone here help me? I am trying to silence e-selectin and ICAM-1 in endothelial cells. I would like to know if this is possible using shRNA

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The adenovirus-based vector AdFoxm1siRNA that expresses Foxm1-specific siRNA was constructed with Invitrogen Block-it Adenoviral RNAi Expression System (Cat. No K4941-00 and V492-20), following the manufacturer’s instructions. A siRNA sequence 5′-GGA CCA CTT CCC TTA CTT T-3 from mouse Foxm1 cDNA was used to design a double-strand DNA that was recombinated into the adenovirus vector. The constructions of human FOXM1-expression adenovirus AdFOXM1 and control virus AdLacZ or AdGFP were described previously (34). The large-scale adenovirus purification and viral infections were carried out as previously described (35). For siRNA treatment, mOct4 siRNA (sc-36124) and control siRNA (sc-37007) were purchased from Santa Cruz. The siRNA transfection was performed according to the manufacturer’s instructions.
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