shRNA

Suspension Stable Cells

General Protocol for Creation of Suspension Stable Cells Using MISSION® Lentiviral Particles (SHVRS)

Protocol provided by Sigma MISSION® RNAi team

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Introduction

The power of the lentiviral delivery system lies in the ability to create stable cell lines from both dividing and non-dividing cells. The lentiviral particles delivery their payload into the target cells, and then, the contents are integrated at high efficiency into the host genome. By selecting for stable integrants utilizing a selectable marker, such as puromycin resistance, a population of successfully transduced cells can be found.  This protocol provides a general method for stably transducing suspension cells in a 96-well plate format. This method can be enhanced utilizing the ExpressMag™ transduction system.  Prior to starting the transduction experiment you must:

  1. Perform a titration of lentivirus to determine the most efficient MOI to achieve high transduction efficiency.
  2. Perform Antibiotic Kill Curve Assay to determine the most efficient concentration of puromycin to achieve the complete selection of transduced cells.

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Materials

  • MISSION lentivirus targeting your gene of interest
  • Polybrene (H9268)
  • Cells (in log growth and at 50% confluence on the day of transfection)
  • Puromycin (P9620) or G418 (A1720) for selection
  • Media
  • Tissue culture incubator—37 °C, 5% CO2, 100% relative humidity
  • Table top centrifuge with 96-well plate adapter and temperature control

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Protocol

Day 0

 0.1   Prepare media containing polybrene such that after dilution of suspension cells the final concentration of polybrene is 8 µg/ml.
 0.2   Seed cells at appropriate density in 96-well (100 µl per well) in media containing polybrene.
 0.3   Add shRNA lentiviral particles to the cells.
 0.4   Centrifuge cells at 800 x g for 30 minutes at 32 °C.
 0.5   Change media immediately following transduction. Remove media and replace with 100 µl fresh growth media (no polybrene).
 0.6   Incubate cells overnight (37 °C, 5% CO2).

Day 1+

 1.1   Remove media 24 hours post-infection and replace with 100 µl fresh growth media containing puromycin. (The concentration of puromycin is determined in Antibiotic Kill Curve Assay).
 1.2   Maintain cells in complete media containing puromycin until they have emerged from the selection and the control non-transduced cells have died.

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Tips

Day 0

  • The growth rates of cells vary greatly. Adjust the number of cells plated to accommodate a confluency of 70% upon transduction.
  • Calculate initial polybrene concentration as: [polybrene] = 8 µg/ml/X where X = 1-dilution factor.
  • Make sure cells are not sensitive to polybrene. Include the polybrene control well in the experiment.

Day 1+

  • Incomplete media removal will affect concentration of the puromycin solution.
  • Use a concentration of puromycin determined by the Antibiotic Kill Curve.
  • Some cells will show decreased viability in the presence of Puromycin. Let the cells recover for 24-48 hours in complete media without antibiotic and then introduce Puromycin.

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