Hi. I'm establishing the cell lines SW948 and LS174T with lentivirus that carry the gene with puromycin resistance (no fluorescence tag). I performed the transduction with polybrene and selected the infected cells with the selection marker puromycin. Two days after starting the puromycin selection the cells start to die.
I already performed different approaches:
- Since I don't know the lentiviral titer, I tried different volumes (5ul-20ul).
- Performed the transduction with polybrene, changed the media 24h later and started immediately puromycin selection.
- Performed transduction with polybrene, centrifuged the cells 90 min at 37ºC, changed the media and let the cells stabilize 48h before starting the puromycin selection. During such 48h without selection, the cells were ok, so it seems that the viruses are not toxic to them.
Before starting those experiments I performed a puromycin curve and I selected the minor concentration to kill the cells in 7 days: 0.75 ug/ml for SW948 and 1.5ug/ml for LS174T.
Did anyone performed a similar work with this cell lines that can give me suggestions to overcome this issue please?
Many thanks!
My first inclination is that since you do not know the titer of the virus, 5-20ul contains little to no virus since all of your cells are dying under selection. I would suggest using a higher volume of virus such as 100ul, 200ul, etc.
Other suggestions:
1. After transduction, you should let that sit for 48 hours and then change the media. By that time the virus should have infected & integrated.
2. After integration, it takes a few days for gene expression from the viral genome (i.e. puro resistance) to kick in. Perhaps wait another 48 hours before starting the puro selection.
3. If you have a GFP lentivirus, I would suggest using it along with your other lentiviral constructs such that you can monitor when GFP expression starts and therefore have an idea when to start the puro selection.
Good luck!
Thank you very much for your kind suggestions. I will try another experiment, with additional time before puro selection and using another lentivirus with fluorescent tag.
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