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pLV-RNAi Protocols – Lenti-shRNA Transduction

Lentiviral transduction for RNAi analysis.

Day 0: Seed cells at appropriate density.

Suggestion: Plate cells so that cell density will be ~10-25% confluent at the time of transduction.

 

Day 1: Transduction. Remove the medium from the tissue culture plate by aspiration and replace it with fresh complete medium containing 5-8 µg/ml polybrene. Gently mix the lentivirus with pipette tip, and add an appropriate amount of virus to each well.

Note: (1) Polybrene may be toxic to some cell lines. (2) The non-concentrated and non-purified lentiviral stock collected from 293T supernatant may contain substances affecting the target cell growth, especially when a large volume of low-titer lentiviral stock is added. In case the lentiviral stock inhibits cell growth, concentration and purification of the viruses may be required. (See below for a protocol for virus concentration) Alternatively, incubation time for transduction can be shortened to hours. For example, the virus-containing medium may be replaced with fresh medium after one hour of transduction.
Suggestion: Transduce cells at multiplicity of infection (MOI) = 1, 5, 10, 25, and 50 to determine the optimal knockdown condition. Spin transduction (1,000 × g for 30-60 min at room temperature) helps increase of transduction efficiency.

 

Day 2: Replace the transduction medium with fresh complete medium to remove lentivirus and polybrene.

Day 3-4: Select transduced cells (>50% confluence is recommended) with medium containing appropriate antibiotics or by flow cytometry to sort out fluorescence-positive cells if necessary.

Note: The optimal antibiotic concentration varies from cell line to cell line.
Suggestion: A pilot experiment should be performed to determine the minimum concentration of antibiotic required to kill the untransduced cells before this experiment

 

Day 6+: Analysis of transduced cells.

Suggestion: Expand the culture of cell lines stably expressing shRNA and store the cell line stocks in liquid nitrogen before analyzing the cells.

 

Day 6-10: Doxycycline is required for inducing gene silencing in cells transduced with the inducible pLV-RNAi vector system. Make a solution of 1 mg/ml in water. Filter sterilize, aliquot, and store at -20° C. To induce shRNA expression, adding Doxycycline to a final concentration of 1 µg/ml in complete medium is recommended. If desired, you may vary the concentration of Doxycycline from 0.01 to 1 µg/ml to modulate RNAi effect.

Note: Some basal expression of shRNA may be observed if the FBS contains tetracycline.

 

Optional: Concentration of lentivirus by ultracentrifugation

  • 1. Filter lentivirus through a 0.45 µm filter.
  • 2. Centrifuge at 25,000 rpm for 90 minutes in a SW-28 or SW-41 rotor.
  • 3. Discard the supernatant and use a Pasteur pipette with an attached P100 tip to carefully remove the remaining medium.
  • 4. Gently resuspend viral pellet in 1/100 volume of DMEM. Let the virus suspension sit for overnight at 4°C.
  • 5. On the following day, mix gently, aliquot and freeze virus.

Optional: Validation of effective shRNA by reporter assay
For validating effective shRNA via a reporter assay, please visit the following web sites for further information:
www.promega.com (psiCHECK vector system)
www.invitrogen.com (BLOCK-iT RNAi targeting system)
www.clontech.com (ProLabel Detection system)