lentivirus question

[panc@k3]

so my sister's got this lab project, and she's trying to do some lentivirus transduction with her WTC11-dCas9-KRAB cells.

Her big goal is to make a stable knockout line, you know, where she basically turns off certain genes permanently.

She's trying to find a good method for it. Does anyone here have any recommended protocols for doing lentiviral transduction with those specific WTC11 cells?

Please share any tips or step by step guides you might have! Thanks a bunch!

 

[Angelina.Mc]

Spinfection helps a lot...try 800–1,000g for 90 mins with polybrene (around 8 µg/mL) and let them recover overnight before selection

 

[nucLe01]

so my sister's got this lab project, and she's trying to do some lentivirus transduction with her WTC11-dCas9-KRAB cells.

Her big goal is to make a stable knockout line, you know, where she basically turns off certain genes permanently.

She's trying to find a good method for it. Does anyone here have any recommended protocols for doing lentiviral transduction with those specific WTC11 cells?

Please share any tips or step by step guides you might have! Thanks a bunch!

That's a cool project!
The dCas9-KRAB system is perfect for stable gene knockdown or repression rather than a true knockout, which is a key distinction.
I'll try to summarize the protocol here:
Day 0 is for seeding and transduction.
Plate WTC11 cells so they're about 70% confluent the next day.
Add lentivirus with the sgRNA and a transduction enhancer like Polybrene (~8 µg/mL).
After 24 hours (day 1), replace the virus-containing media with fresh media
Day 2, you begin the antibiotic selection.
The virus likely has a Puromycin resistance gene, so add Puromycin to the media, but first she should run a kill curve.
I think a starting dose of 0.5-1 µg/mL is a good guess for WTC11s.
Then days 3 to 7, continue changing the media with fresh antibiotic every few days until a healthy, resistant population of cells is established.
And just a final tip, make sure that viral titer is high and she optimizes the MOI.
She can verify repression with RT-qPCR or a Western blot!