shRNA gene silencing Mouse - R221a IL4Rα

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.

Start discussion

Found 1 discussion for this experiment


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

Share your thoughts or question with experts in your field by adding a discussion!

Found 1 matching solution for this experiment

Protocol tips
PyVT-R221a cells were isolated from a polyoma middle T oncoprotein (PyMT) tumor as previously described [13]. The R221a line was authenticated by expression of the polyoma antigen, and the morphology and growth rates of both cell lines were consistently monitored. Cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS, Atlanta Biologicals) at 37°C with 5% CO2. Control non-target and specific murine IL4Ra-targeted shRNA lentiviral particles (Santa Cruz Biotechnologies) were used to infect R221a cells. Post-infection, shRNA-expressing cells were selected with puromycin dihydrochloride (Sigma). The percent of IL4Rα protein knockdown was calculated by densitometry in Adobe Photoshop® following western blot analysis.
Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms