I am trying to transiently co-transfect dCas9, gRNAs, and a puromycin-resistance plasmid into A549 cells using Lipofectamine 3000, but I see almost complete cell death by 4 days post-transfection when I need to collect for ChIP. This is a control. I hope to eventually use epi-dCas9 constructs, if I can get the control cells to live!
I am transfecting into 70% confluent 10-cm tissue culture dishes. On the day of transfection, the cells look as expected. 12 hours after transfecting I replace the medium with fresh F-12K/10%FBS/1%PS. When I come back 24 hours after transfection to add the selective marker, the transfected cells have already started to die. I've noticed that the cells around the perimeter of the dish are very sparse and they start to look very elongated and dark. The nuclei look darker as well. I notice dead cells and dark dots floating the medium.
I have already optimized the amount of puromycin (1.5ug/mL) and the amount of puromycin-resistance plasmid (1.25 ug pBABE-puro). When I only transfect pBABE-puro, I do not see any cell death with or without selection. When I treat WT A549 cells with 1.5ug/mL puromycin, they totally die after 4 days, as expected. Therefore, I believe that my transfection technique and puromycin conditions are working.
I have tried adding only transfection reagents, and the amount of Opti-MEM and Lipofectamine 3000 reagents I am using do not affect cell viability.
At this point, it seems like the dCas9 or gRNA plasmids are killing the cells. However, I contacted the authors of the study in which these constructs were designed and they did not experience this issue. I prepared my plasmids using Thermo Scientific GeneJET Maxi Prep Kit, which results in ~300 ng/uL plasmid DNA. I know that this concentration is low, but I haven't been able to successfully get higher concentrations (any suggestions for this issue?). I've read that large CRISPR-dCas9 plasmids are difficult to transfect because of their size, but other studies report using these constructs without issues.
I'm at a loss for what could be going wrong! Has anybody experienced this issue or have suggestions of what I can try?
Thanks in advance for your help :)
It seems that the plasmid DNA quality is poor. The concentration of plasmid is too low for maxiprep. Maybe try another isolation kit to see if it helps?
Another factor often is overlooked for plasmid DNA prep is that it may contain too much endotoxin, which often results in excessive cell death.
Chengkang Zhang The kit I am using does include an endotoxin-binding reagent, but I agree that the concentration is low. Do you have a favorite maxi prep kit for transfections?
Commercial maxi prep kits are not that different. Have you done miniprep and how much DNA did you get from a mini prep? If the yield is good from a miniprep, simply scaling out by doing many minipreps and pool the DNA together might solve your problem.
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