Hello,
I was just wondering if anyone had done shRNA with lentivirus in HEK 293 successfully and if so, what concentration of Puromycin was used? I've done a Kill Curve on a 6 well plate and I think 0.75µg/mL is appropriate, however I cannot find any papers to back this up. They all say refer back to the original protocol, which actually says to optimise yourself, hence I have no way of backing up that my Kill Curve concentration is correct. I'd be grateful for any advice, thanks.
Dear Sarah,
The killing dose of puromycin vary from cell lines, 0.5-5 ug/mL in common. First, I suggest you use a couple of wells to titer the concentration for each cell line to find the optimal dose. The cells may die after screening for 2-3 days. Second, it works well to screen with puromycin when passaging. Third, you say that your virus is not able to infect the cells or the resistance is hardly expressed. I think, the reason may be that the titer of your virus is not enough to infect your cells or the MOI is not suitable.
Genemedi got a rich experience in lentivirus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
Hi Sarah, you're concentration sounds about right for many cell types. However, I've never used Puro for 293 myself, only various cancer cells. As for the 'right concentration,' in my opinion there is no right concentration. If there was, it would be what is killing all of your cells in the time-frame you require. Other people doing the assay might have a slightly different concentration of Puro for various reasons, cells that have been treated differently, cells at different confluence, or various other factors that make their concentration right for them but which wouldn't be right for you. As long as your cell line is stably transfected and you can show it by various means, and you make at least two different pools or single cell clones for each stably transfected cDNA or shRNA, I don't think they would question whether you're Puro concentration was excessive or too dilute, unless of course your kill curve was complete in two days, but that is another topic.
Why u want any backing from literature? Repeat couple of times growth curve assay in presence of different concentrations of Puromycin. It will gives you the lethal dose for HEK293.
Depending on your cell batch and status of your cells, the concentration will allways vary a bit - this is why you have to test it out to get the optimum concentration - and even when thawing a new batch of cells, in principle you have to validate if your concentration is still valid.
As you obviously already have a concentration - go ahead with it, and keep in mind, with a new batch the concentration might change a bit.
Erik Martin is completely right. The right selective pressure of antibiotic is dependent on so many aspects, that what you got from exprimental killing curve is right for your cells in your medium and serum and for your puromycin stock. For my cancer cells I was using 2 ug/ml of puromycin and my student was using 1.8 ug/ml as his treatment of our tumor cells was a little more "drastic" so they were a little more sensitive to antibiotic.
Although the question is rather old, as a guide here is a list by Thermo Fisher for selecting puromycin concentrations ideal for different cell lines:
https://www.thermofisher.com/de/de/home/life-science/cell-culture/transfection/selection/puromycin.html
They did not mention the different pre-treatments, however as a general guidance I think it is useful. ;-)
Hi Sarah,
We use 0.4 ug/ml (final) of Puromycin for 293T, but for other cells it might be different. For example, I have done it for THP-1 (200,000 cells/well) and OCI-AML3 (150,000 cells/well), both in 12-well plate. The cells were treated with 0.25, 0.5, 1, 2, 4, 6, 8, & 10 ug/ml (Final) in duplicates. We found that (over one week), the lowest concentration (0.25 ug/ml final) might be the best choice. Even 0.5 ug/ml was pretty toxic for the cells.
Best,
Reza
Dear Sarah,
The killing dose of puromycin vary from cell lines, 0.5-5 ug/mL in common. First, I suggest you use a couple of wells to titer the concentration for each cell line to find the optimal dose. The cells may die after screening for 2-3 days. Second, it works well to screen with puromycin when passaging. Third, you say that your virus is not able to infect the cells or the resistance is hardly expressed. I think, the reason may be that the titer of your virus is not enough to infect your cells or the MOI is not suitable.
Genemedi got a rich experience in lentivirus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
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