Hi there!
Do you have any recommended protocols for lentiviral transduction of WTC11-dCas9-KRAB cells? I would like to make a stable knockout line.
Thank you!
Hello,
I can give you a very general starting point for your lentiviral transduction but we do not have an official SOP. Also, this is to knockdown gene expression, not create a stable knockout. If you want to really knockout the gene, you would need to generate that for your gene of interest.
Normally, we follow an 8 day experimental timeline similar to below:
Day 0: Harvest cells and seed into wells/dishes. Immediately, follow seeding of cells with addition of lentivirus at an MOI of 0.8. We do not add polybrene. The number of cells will depend on what size dish or plate you are using. The entire transduction is set up with mTESR + Ri without supplement and without Pen Strep. After 6 hours, we add back supplement to 1X concentration.
Day 1: Cells recover and we feed with the normal mTESR+P/S (with supplement +P/S).
Day 2: Split the cells 1:2 on Day 2, and then start puro selection using 0.8ug/mL.
Day 3: Continue selection by feeding cells with 0.8ug/mL puro.
Day 4: Increase puro selection to 1 ug/mL.
Day 5: Split cells again and set up for the assays being run.
Day 8: Analyze/ harvest cells.
All dishes/plates are coated with standard amount of Matrigel.
All of these steps, especially for splitting, may vary depending on how your cells grow.
Hope this helps!