Hi, i performed lentivirus transduction on A549 with MOI 5. 72 hours post transduction, i observed the cell under microscope. Almost 70% cell were successfully transduced. Then, i treat the cell with 1.5ug/ml. puromycin to obtain stable cell line. unfortunately, after 7 days treatment, only 70-80% cell express GFP. I expect the puromycin will kill the non-transduced cell since the control well (cell without transduction) showed that all cells were died on 5th day of treatment. Could someone suggest to me what should i do to obtain 100% GFP-expressing cell?
I agree with Hyde Chen. Passaging the cells could help. You could also try increasing your puro concentration. 5ug/ml is a widely used concentration, and we have used it on A549 in our lab. If you have access to a cell sorter, I would suggest using that as well after increasing puro concentration for a few days.
I see you have received some good suggestions already. I always isolate single cells to obtain pure clones after transfection. For this, you can use a cell sorter (as Danielle May suggested) or limiting dilution.
Increasing puro concentration may help as well, although I would titrate using untransfected cells first. Lot to lot variations and other factors may cause differences in efficacy.
Good luck!
Tq hyde chan for your suggestion. i dont do passaging during puromycin selection but only do that after 7 days of selection. I already freeze down the cell for storage. Is it possible if i grow the cell and do the selection again?
tq danielle may for your suggestion. i try concentration 1.5ug/ml because all control cell without transduction died with this transduction. But as u suggest, sorting GFP expressing cell will be a good way
tq wail hassan. i already done titration with different concentration of puromycin on A549 cell. I found that 1.5ug/ml is the best concentration to kill all cell within 3-7 days
Noor,
The answer to your question is yes. We freeze cells and thaw them later to complete clone purification all the time.
The only question I have is whether your intention is to obtain clones that resulted from independent integration events. When you transfect, the DNA remains in the cytoplasm for a while, but as cells propagate, DNA integrates somewhere into one of the chromosomes. This is a rare event as I am sure you know. The question is whether the transgene integration disrupts an endogenous gene or other important sequences. For this reason, some investigators aim to isolate clones that have resulted from separate integration events to use later for verification of any results you obtain using any of your clones. I hope this is clear, but if not, I am more than happy to explain in more detail.
So, long story short, freezing and passaging do not allow for obtaining independent clones, since all cells get mixed together. A colony that remains after antibiotic treatment is widely regarded as an independent clone. So, that is all you lose when you freeze.
Hope this helps!
Best!
hi wail, i'm sorry cause i dont really understand your explanation. in my case, i used lentiviral in other to get stable transfection, which is the transgenes are integrated into the genome. when i performed transduction of cell with lentiviral, my intention is to obtain as much as possible cell that were successfully transduced with lentiviral that carrying the transgene
When you transfect (with lentivirus or just a plasmid DNA), the transfected DNA will eventually integrate into the genome of the transfected cells. The location in the genome where the introduced DNA integrates is random.
Let's say you want to test whether introducing gene X into the cells will make them sick. If you transfect your cells with gene X, and they become sick, you would have one of two possibilities: 1) gene X made the cell sick or 2) when gene X integrated into the genome, it disrupted an important gene causing the cells to become sick.
To resolve this issue, researchers like to generate multiple cell lines that are stably transfected and have been generated by independent integration events. This means those cell lines have your gene X integrated in a different location in each one of them. If all the cells show the same phenotype (in this example become sick), then you should have more confidence that gene X is responsible for this phenotype.
How to generate cell lines from independent integration events? Essentially what you did without passaging or freezing. After you infect the cells with the lentivirus and add the antibiotic, many cells will dye leaving behind transfected cells. Usually, transfected cells will be far apart from each other and as they propagate, they will form colonies. Each of those colonies is a clone.
Hope this helps!
Best!
Wail
tq so much wail for the very detail and good information. i really appreciate it
Hi,
You must remember that death/dying cells gives strong false positive results too.
If you need pure culture you should sort cells with the sorter.
Good luck!
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