shRNA

Spinoculation Protocol

The MISSION® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. This protocol describes the use of MISSION TRC shRNA Lentiviral Particles particularly for long term silencing and phenotypic observation in suspension cells. The following protocol has been developed from the literature1 for use in a 6-well plate with MISSION lentiviral particles.

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The ExpressMag™ system (SHM01 or SHM02) has also been optimized to transduce suspension cells in 24 hours less time than this protocol and in multiple tissue culture formats.

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Materials

MISSION lentiviral particles

Utilize SHC002V or SHC001V for optimizing transduction protocol. Transduction of SHC003V can be monitored via FACS or fluorescence microscopy, but protocols for this are outside of the scope of this document. It is critical to first optimize your protocol, so that you are confident in your transduction efficiency.This will deconvolute effects of the lentiviral payload (either shRNA or a custom payload) from transduction efficiency. Then, when switching to a MISSION lentivirus that expresses an shRNA, any changes in the resultant number of colonies or viable cells can be attributed to effects of the shRNA and cannot be attributable to a low transduction efficiency. After transduction efficiency has been optimized, utilize MISSION lentiviral particles expressing shRNA targeting your gene of interest or utilize a custom MISSION lentiviral particle expressing any shRNA or gene of interest.

Puromycin (P9620)
10 mg/mL stock solution for selection

Full Media
For Jurkat cells DME including 10% heat inactivated fetal bovine serum or media for your cell type.

Trypan Blue (93595)

12 x 15 mL Conicals (CLS430790)

6-Well Tissue Culture Plate (CLS3516)

Swinging Bucket Centrifuge
The centrifuge should be equipped with temperature control and prewarmed to 32 ºC prior to the cell transduction.

Hemacytometer (Z359629)
Hemacytometer is used for cell counting.

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Protocol

  1. Determine the final puromycin concentration to be used in the selection. The exact concentration is cell line dependent and should be determined by a 72 hour cytotoxicity assay on non-transduced cells.
  2. Utilize only sterile items and work using standard tissue culture sterility techniques throughout this procedure.
  3. Pre-warm all media to 37 °C.
  4. Count cultured viable Jurkat cells.
  5. Dilute Jurkat cells into complete medium to a final concentration of 1x105 cells/mL with a final volume of 14 mL.
  6. Transfer 2 mL of 1x105 cells/mL into each of six sterile 15 mL conical tubes.
  7. Add MISSION lentiviral particle solution to the Jurkat cells such that the final multiplicity of infection (MOI) equals 0, 0.1, 0.5, 1.0, 5.0, or 10 per conical. The 0 MOI represents your non-transduced negative control.
  8. Centrifuge the cells at 800 x g for 30 minutes at 32 °C.
  9. Virus containing medium is aspirated and disposed of appropriately as per your institution.
  10. Each cell pellet is resuspended in 2 mL of media by gently pipetting the pellet up and down, and each resuspended pellet is transferred to its own well in a 6-well plate tissue culture plate.
  11. The plate is returned to the tissue culture incubator for three days.
  12. After 3 days, transfer cells to 6 separate 15 ml conical tubes and pellet at 200 x g for 5 min. Remove media and replace with 2 ml complete media containing puromycin.
  13. Return tissue culture plate to incubator overnight.
  14. Cells are maintained in puromycin until the non-transduced negative control cells have died as determined by inspection or Trypan Blue staining.

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Notes

Use the equation below to calculate the MOI in a well. The ViralTiter will be provided in your certificate of analysis from Sigma. The VolumeofVirus is the volume in mL that is added to the well/centrifuge tube containing you the cells to be transduced. The NumberofCells is equal to the total number of viable cells in the well/centrifuge tube.

References

  1. O'Doherty, U.; Swiggard, W. J.; Malim, M. H. J Virol 2000, 74, 10074-10080.

 

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ExpressMag is a trademark of Sigma-Aldrich Co. LLC

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