I am trying to knockdown a protein in Jurkat cells by shRNA lentiviral transduction. I prepare virus particles in HEK-293T cells using shRNA construct + psPAX2 + pMD2.G in the ratio given on addgene's site. It has worked before very well. But now there are many problems. I add HEK-293T virus supernatant into 2 million Jurkat cells in a 1:1 ratio (1 ml of virus supernatant + 1 ml of fresh media). After 24 hours, I spin down the cells and add 2 ml fresh media. 24 hours post that (48 hours after initial infection), I add 1 ml media with puromycin at a concentration of 1 ug/ml (total volume is 3 ml so 3 ug of puromycin) for selection. Cells are healthy until this point. Almost immediately after this, almost all cells die. Within 24 hours, all cells are either dead or undergoing apoptosis. Any help?
Do you have a fluorescent reporter in your shRNA cassette so that you can verify the transduction of the Jurkat cells?
Do you have a non-transduced control included during the puromycin selection? I presume that the concentration of puromycin you are using was determined using a kill curve on these cells, correct?
Double check the concentration of the puromycin solution you are using. Perhaps you are using more than you intend.
Can you increase the volume of viral supernatant used in the transduction? Do you have to mix 1:1 with the fresh media?
John Hardy Lockhart: I don’t have a fluorescent cassette unfortunately. I determined puromycin concentration with a kill curve initially. I can try to increase the viral supernatant and check if that works.
I have also kept transduced cells with out puromycin and there have been times when they also start becoming apoptotic in a week.
I'd recommend double checking your puromycin concentration. At the very least treat some untransduced cells during your antibiotic selection time.
The transduced cells dying after some time is concerning. Could it be an effect of your shRNA? Do you see a similar effect in a non-targeting or scramble shRNA control?
I don’t think it’s an effect of knocking down my protein since I see it with different types of shrna, even the scrambled one. I will definitely be rechecking the puromycin concentration. On the other hand, once when I had previously done this, I probed for p24 antibody by running the protein lysate of these cells in a western. I got p24 bands. So I am partly reassured that virus is being made. If you or anyone else can think of anything else, please let me know, thanks in advance!
I add 500uL of viral media to total 3mL (2.5ml cells media + 0.5ml viral media), I work with Brain tumor stem cells so they are sensitive to viral media (serum containing DMEM). So maybe check whether they are sensitive to viral media as well? Also, I use iFBS containing media it was a bit better in terms of transfection of HEK cells compared to FBS. Even my initial transfection the BTICs cells were dying during puromycin media change, but now with same puromycin concentration (3ug/ml) cells are surviving and able to passage until P5 before protein levels come back.
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