stable transfection problem - (Apr/13/2010 )
hi, everyone. I have been trying to use BLOCK-iT miRNA expression vector to knockdown a gene in a T cell line. This cell line is virus-transformed and hard to transfect. So I used electroporation by Neon. the big problem is that most cells died after 24h and at 48 or 72h, there were almost no living cells. Is it possible for the rest living cells to grow and even to be selected? if its impossible, then what survival rate should I get before doing selection? Thanks in advance.
Did you try using different concentrations of your vector? It might be that your cells suffer from DNA "poisoning". You might want to setup an experiment in which you electroporate with the empty vector as a negative control, so you know whether your cells die from the electroporation or from DNA poisoning or whether they die from the miRNA you use...