shRNA gene silencing Mouse - FL83B HtrA2

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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5 years ago

5 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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HTRA2 MISSION shRNA Lentiviral Transduction Particles HtrA serine peptidase 2

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	To establish a stable HtrA2/Omi-depleted cell line, FL83B cells were infected with a mouse HtrA2/Omi specific shRNA-encoded lentivirus (Sigma; SHCLNV-NM_019752). An shRNA negative control lentiviral particle (LV-Control) was used as a negative control. To generate a stable cell line, FL83B cells were plated at a density of 1 × 105 cells per 60-mm culture dish and infected overnight with five multiplicities of infection (MOI) lentiviral particles in the presence of 8 μg/mL hexadimethrine bromide (Sigma). After infection, the transduced cells were selected using 10 mg/mL puromycin (Sigma) for 2 weeks and incubated at 37 °C in a humidified incubator with 5% CO2. Suppression of HtrA2/Omi expression in selected cells was confirmed by western blot analysis.

Protocol tips
To establish a stable HtrA2/Omi-depleted cell line, FL83B cells were infected with a mouse HtrA2/Omi specific shRNA-encoded lentivirus (Sigma; SHCLNV-NM_019752). An shRNA negative control lentiviral particle (LV-Control) was used as a negative control. To generate a stable cell line, FL83B cells were plated at a density of 1 × 105 cells per 60-mm culture dish and infected overnight with five multiplicities of infection (MOI) lentiviral particles in the presence of 8 μg/mL hexadimethrine bromide (Sigma). After infection, the transduced cells were selected using 10 mg/mL puromycin (Sigma) for 2 weeks and incubated at 37 °C in a humidified incubator with 5% CO2. Suppression of HtrA2/Omi expression in selected cells was confirmed by western blot analysis.

Protocol tips

To establish a stable HtrA2/Omi-depleted cell line, FL83B cells were infected with a mouse HtrA2/Omi specific shRNA-encoded lentivirus (Sigma; SHCLNV-NM_019752). An shRNA negative control lentiviral particle (LV-Control) was used as a negative control. To generate a stable cell line, FL83B cells were plated at a density of 1 × 105 cells per 60-mm culture dish and infected overnight with five multiplicities of infection (MOI) lentiviral particles in the presence of 8 μg/mL hexadimethrine bromide (Sigma). After infection, the transduced cells were selected using 10 mg/mL puromycin (Sigma) for 2 weeks and incubated at 37 °C in a humidified incubator with 5% CO2. Suppression of HtrA2/Omi expression in selected cells was confirmed by western blot analysis.

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