Hi,

did you perform a kill curve upfront? 2µg/ml sounds quite high for me. In addition I alwys passaged the cells after 72h and then started Puro selection to give them some time to recover form the transduction

Sven

Hi Sven Reister and Connor E Woolley

Thanks for your suggestions. I did a kill curve and it was a toss up between 1 ug/mL and 2. I went with 2 following other's suggestions and also as I've seen the same concentration used for lentiviral transductions of the same cells in numerous papers. Furthermore, cells were allowed to recover for 48 h and grow to 70-80% confluence before beginning selection. My plan was to maintain the puromycin at 2 ug/mL until a stable polyclonal population was selected and expanded and then reduce to 1 or 0.5 for maintenance. I suppose there will be a a threshold above which the resistance transgene will not be able to maintain the antibiotic resistance, particularly under the stress of trypsinisation? I'll definitely go with your suggestion and reduce the dose as 1 ug/mL will kill all non-transduced cells within 5-7 days. Thanks for your help really appreciate it!

Hi Eoin Dervan ,

Did you manage to get a stable population of silenced cells? I'm facing a somewhat similar issue with a mouse adherent cell line Gc1-spg in which I'm trying to integrate an inducible silencing shRNA plasmid without GFP.

Strangely, my cells survive for 7-10 days with puromycin (2ug/ml as reported in publications and based on kill-curve), form colonies and then die. I tried reducing the concentration after 7 days to 0.5 ug/ml but with the same outcome. I notice the same behaviour irrespective of whether I transfect the shRNA plasmid into the cells with Lipofectamine or I infect the cells with lentiviral particles. Somebody suggested that the cells could be dying due to puromycin toxicity and that I select with puromycin (a higher concentration than 2ug/ml) for only 72 hours, wash the cells and continue to grow them without any selection and then screen for silencing. While I plan to give that a try, I look forward to hearing about how things worked out for you.

Hi Bhavana Kayyar ,

I did manage to get stable populations. My transduction efficiency appeared >90% by GFP expression, the majority were surviving selection, and all negative control cells were dead within 4 days. My issue was they were then dying after trypsinisation. I removed the selection pressure when trypsinising and then allowed the cells to adhere and recover before adding back in the antibiotic. Do you have a fluorescent reporter in your construct? Rather than select with antibiotic could you do cell sorting? What gene are you knocking down? The gene could be essential for cell survival and may be that the knockdown itself is toxic? I think trying the shorter selection time and then allowing the surviving cells to expand is definitely worth a try as you mentioned. Even if your population isn't 100% "pure", if your knockdown is sufficient you could proceed with that population. You could then select intermittently with antibiotic to ensure the population isn't taken over by non-transduced cells (particularly if cells expressing higher levels of your target gene have a growth/survival advantage). I'm far from an expert but hope my comments are of some use. Best of luck and feel free to message again if I can be of any help!

Hi Eoin Dervan ,

Thanks for the details. Unfortunately, I do not have a fluorescent reporter in the construct and so sorting is not an option. I know that the gene is not essential for cell survival since we have ben able to knockdown this intronic lncRNA previously with siRNA and with Wnt cue and besides, the constructs are for inducible silencing and the cells die way before I can induce expression of the short hairpin. I can't use siRNA or Wnt cue now since I need a very homogenous population of cells .Thanks again for the suggestions. Hopefully, intermittent antibiotic selection will work for me.