Melanoma cells transfected with lentiviral shRNAs, which were nicely adherent before freezing down, have transformed into suspension cells with reduced viability after thawing. Even non-target control shRNA transfected cells appears sick and in suspension after thawing so it does not appear to be transfection specific effect.
I would say that this is unusual and probably not down to the lentivirus. I could be wrong of course, but our lab have successfully used lentiviral shRNAs in melanoma cells.
Could I ask 3 things:
Where did you source your lentivirus?
Are the cells you are using established cell lines?
What do you freeze your cells in? (although I guess you have no problem thawing out 'wildtype' cells?)
I would check the media formulation, I been working with the lentiviral shRNA system and the lentiviral control particle and never had that problem.
Good luck.
First of all you talk about transduction, not transfection, correct? So you have a stably integrated cell line for both shRNA and control shRNA and was freezing them afterwards? Are the original cells behaving similar when you freeze and thaw them at the same timepoint?
It might be a passage problem, I've seen detachment happening in HEK 293T fibroblasts at a passage number reproducible (each time I reached that number the suddenly came off).
Whatever your target is, are you sure your control is not toxic? Did you use an established control like luciferase or GFP shRNA or a scrambled? The later one have often significant side effects.
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