shRNA gene silencing Mouse - CT26 OPN

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

Discussion

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

AM5770: pSilencer™ 3.1-H1 neo

Thermo Fisher Scientific

Protocol tips
CT26 murine colon carcinoma cells, syngeneic to BALB/C mice, were grown as monolayer cultures in DMEM–10% FBS (Invitrogen, Carlsbad, CA) supplemented with 100 IU/ml Penicillin and 100 µg/ml Streptomycin. Cells were maintained in a 37°C incubator with 5% CO 2 humidified air. Cell lines that were stably transfected with plasmid vectors were maintained in DMEM–10% FBS supplemented with 100 IU/ml Penicillin, 100 µg/ml Streptomycin and 750 µg/ml Geneticin (Invitrogen).Using GenBank™ sequence NM 009263 for murine OPN cDNA and computer analysis software developed by Ambion, Inc., we selected two candidate sequences in the OPN cDNA sequence for RNAi ( Figure 1A ). These 21-nt sequences showed no homology with other known mouse genes. The corresponding sense and antisense siRNA sequences for each target are also shown ( Figure 1A ). Synthetic, annealed, siRNA oligonucleotides were synthesized chemically and gel-electrophoresis purified (Ambion, Austin, TX) and used during transient transfection experiments. Murine mismatch or scrambled siRNA sequences (Ambion) possessing limited homology to mouse genes served as a negative control. For stable RNAi we designed a hairpin siRNA sequence that contains both sense and antisense siRNA sequences against OPN target 2 and flanking BamH1 and HindIII sites ( Figure 1E ). This sequence was chemically synthesized and PAGE-purified (Sigma-Genosys, The Woodlands, TX) and cloned into pSilencer neo™, an expression vector containing an H1 RNA polymerase III promoter ( 24 ) and a neomycin antibiotic resistance gene (Ambion). A pSilencer neo™ vector that expresses mismatch hairpin siRNA with limited homology to mouse genes (Ambion) served as our negative control. Harvesting of CT26 cells using Trypsin was done 24 h prior to transfection, and plated at a density of 5 × 10 5 cells/well in 6-well plates (Costar, Corning Inc., NY) in DMEM–10% FBS without antibiotics. Reconstituted, annealed siRNA against target 1 (sR1) and siRNA against target 2 (sR2) were diluted in OPTIMEM I (Invitrogen) to a final concentration of 50 nM and transiently transfected into CT26 using Lipofectamine 2000 (Invitrogen). The medium was replaced with DMEM–10% FBS after 4 h. CT26 wild-type cells (WT), CT26 incubated with Lipofectamine 2000 alone in the absence of siRNA and CT26 incubated with Lipofectamine 2000 and mismatch siRNA (sR−) were used as controls. Cells were harvested 48 h after transfection and OPN protein levels were quantified by western blot-analysis in triplicate assays.
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