I want to transduce MDA-MB-231 cells with lentivirus in order to achieve a stable expression of MDR1. But unfortunately, after transfection and antibiotic selection, the cells die after they are exposed to the compelet media. I have an idea how to perform the transduction on these cells, I would be very grateful.
Cells should never die in complete media....How long do you select in puromycin? Did you make a puromycin concentration killing curve for your cell line? How do you carry out the transduction process? Do you use any transduction enhancers like polybrene? What is the construct in the LV driven by?
Thanks Khan,
In the first i prepared a serial dilution of hygromycin and got nontoxic concentration and Cells were selected in hygromycin for 5 day. I donot use polybrene because i do this process successfully and without the need for polybrene on another cell!!
in this process in day 1 cells were transfected with LV in different concentration (MOI10 and 5), based on the protocol cells are placed on 4 0C for 2 hours and after this returned to incubator for 24h.
in day 2 the media was replace with compelet RPMI (FBS10% and pen/strep) for 48h.
in day4 the cells were exposed to selected concentration of antibiotic for 5-10 days.
After exposure to antibiotics, MDR1 gene expression should be evaluated, but unfortunatly the cell die!!
If the transduced cells die during selection, you may want to make sure that the cells have been transduced under your conditions. Have you used any reporter gene within your MDR1 Expression construct or another vector on this cell line, in parallel? Otherwise, waiting 4 days for selection seems quite long and may lead to inactivation of selection marker expression, in the meantime - ususally, the selection marker should be sufficiently expressed 48 h after starting the transduction process.
Do you have a control-vector without the gene-of-interest run in parallel? And if so, do the cells transduced with the control-vector die as well? If both die you might have a problem with transduction efficiency. Different cell lines can be harder to transduce, and another cell line might need more/less polybrene. If only the cells transduced with the-gene-of-interest die, and the cells transduced with the control-vector survives, perhaps over-expression of the gene is toxic to the cells? As Michael mentioned above, using a reporter gene is a good idea to ensure the cells are transduced before selection.
As I understand from your messages your lentiviral construct has hygromicin resistance, does your lentiviral integrates or not? Because nowadays you can buy both variants on the market. If it integrates, than it doesn't matter for how long you do selection, if it is not, than 96hours is top, but I would go for 48 - 72 hours of selection.
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