The cells start to die in about 6 days after infections. Surprisingly, the cells more sensitive to the luci shRNA control vector then to the shRNA than to the gene of interest. The empty lentiviral vector is not toxic. Washing away of polibren after infection does not help. Cells are human cHL, B-NHL and B-ALL cell lines.
Thank you in advance.
Have you treated your cells with polybrene only (without control vector)? It may induce cell death during the initial contact and you may be seeing after effects even if it is removed from the media.
They are dying under the selection or without addition of antibiotic? If they are dying after addition of antibiotic, problem might be transduction efficiency. Purity of your plasmid may decrease your virus titer. (because of low trasfection efficiency in your packaging cell). If it starts to die after transduction without addition of antibiotic, no comment without additional information!
Dear Tuncay, the cells were not selected. They just do not proliferate or proliferate slowly. The luciferase shRNA is most toxic. I just designed a scrambled control and is waiting for results. My favorite hypothesis is that shRNA induces antiviral (off-target) response.
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