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.
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.
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.
10 mg/mL stock solution for selection
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 is used for cell counting.
Use the equation below to calculate the MOI in a well. The ViralTiter will be provided in your certificate of analysis. 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.
|MOI||=||Viral Titer (TU/mL) x Volume of Virus (mL)|
|Number of Cells|