Hi. Recenty, we were confused about the problem in microRNA overexpression stable cell line construction. We want to study the gain of function effect of a mircroRNA in HUVECs and breast cancer cells, so we bought a microRNA overexpression lentivirus with puromycin coexpression. Transfected cells was challanged with puromycin for 7 days. 2~ fold of overexpression effect was detected by Q-pcr at the first time, but what confused us is that no overexpression effect could be detected after 1 or 2 passages. We thought the unstable expression of the whole sequence so the cells was screened by puromycin again, but no dead cell was observed. The same problem was arised in 293T cells. We bought another lentivirus from different distributor but the same problem still arised.
FCS or FBS has the tendency to inhibit your degree of transfection and what concentration of FCS do you use in your media? for efficient lentiviral transfection, optimizing the media is very important.
Hi, M.G. Tedla. Thank you for your response. We have altered different concentrations of FBS during transfecting, 10% at first, 1% and even non-serum media was also tried. But the problem can't be solved.
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