What is shRNA lentivirus?

Small hairpin RNA (shRNA) and lentiviral vector RNA interference has been used as a research tool to control the expression of specific genes in numerous experimental organisms and has potential as a therapeutic strategy to reduce the expression of problem genes[1,2].

How much polybrene do I add?

Make sure to use the polybrene-containing media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute 350,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or inverting the tube.

Can you transduce cells twice?

It is a routine practice in our lab (human and murine cells) and we have no problem with this type of protocol. You can also do the transuction in two times if you want an offset expression of the two transgenes, it depends of the experimental purposes.

What is MOI for lentivirus?

MOI is the ratio of the number of transducing lentiviral particles to the number of cells. It is highly recommended that for each new cell type to be transduced, a range of MOI be tested. Plate 1.6 x 104 cells into wells of a 96-well plate with 120 µL fresh media. Add control virus to cells in a range of MOIs.

How does shRNA lentivirus work?

Lentiviral Vectors Enable Permanent Knockdown of Targets The shRNA becomes stably integrated into the host cell genome. As cells divide, the shRNA is passed on to daughter cells. Using lentiviral vectors for expression of shRNAs provides permanent knockdown without needing to transfect the cells multiple times.

How do you transfect shRNA?

Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature. Wash the cells twice with 2 ml of shRNA Transfection Medium: sc-108062.

Why polybrene is used for transduction?

Cationic polymers, such as polybrene and protamine sulfate, are typically used to increase the efficiency of retrovirus-mediated gene transfer, however, the mechanism of their enhancement of transduction has remained unclear.

Is polybrene toxic to cells?

Polybrene is considered non-toxic at low concentrations, but has been found to negatively affect cell proliferation in some cell types, such as keratinocytes, at concentrations greater than 10 µg/mL [16].

How long can lentivirus be stored?

Since Lentiviruses are sensitive to freeze-thawing and the titer drops with repeated freeze-thawing, aliquot viral stock should be stored at – 80°C freezer immediately upon arrival for long-term usage. While virus titer redetection is suggested before using if the lentiviruses have been stored for more than 12 months.

How long is polybrene good for?

In your case, you should make a stock solution at 6mg/mL so that it’s an easy 1:1000 dilution when you are adding to your cells. Polybrene is stable for 2 months at 4 degC but it’s best to aliquot the rest and freeze at -20 degC.