I am doing primary fibroblast cell culture and transduced my cells with a lentivirus with 10^7 TU/mL. I added 7.5 uL of virus to about 250,000 cells to have an MOI of about .3. Of note polybrene was also added at 8uL/mL. When the antibiotic kill curve was done all of the control cells dies but only 1 percent of my transduced cells died. I understand that adding polybrene will increase my transduction efficiency, and given that the 10^7 TU/mL number was generated without polybrene, my actual TU/mL might be higher when used with polybrene. Regardless, I don't think polybrene would change my transduction efficiency from about 25-30% (MOI .3) to 99%. Additionally, my cultures had odd morphologies and a reduction in proliferation that are not expected to arise from my vector (just a promoter-antibiotic resistance-T2A-dCas9).
Does anyone know what the issue might be?
Thanks!
Hi Michael Fitzgerald
Which drug are you using for selection, at which concentration and how long has the exposure been?
In my hands it was harder to get a clean selection with G418 as post-transduction my cells seemed to be resilient to this drug and prolonged exposure is known to induce resistance.
Upon switching to Puromycin at 2ug/mL, I had better results (I had constructs for both these selection markers).
Best
Aparajita
change the condition of cell culture add more growth factors (serum), and use m. o. i 0.01 for 3 or 4 passages...
Hi, when you have lots of resistant clones they degrade the antibiotics so the concentration is lowered and non transduced (non resistant) cells can survive (this effectively happen easily with G418). It is also possible that during selection the resistant cells proliferate and fill the plate: how do you calculate your 1% ?
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