Hi!
I am trying to knockdown AR protein in a prostate cancer cell-line and I have used a commercial Lentiviral shRNA particles (pLKO.1 plasmid Titre 10^6). Briefly, I seeded 20,000 cells per well in a 48 well plate and kept them overnight to adhere. Next day, I added the particles at 2-5-10ul concentrations along with 8ug/ml Hexadimethrine bromide (not lethal for the cells) in different wells and incubated them for 48 hours in culture conditions.
Post-incubation, fresh culture media without antibiotic was added for 12 hours. Then, 0.5ug/ml Puromycine was added, which killed all the cell in non-transduced well of the same plate, but not the transduced cells (with all conc. 2-5-10ul). I increased the concentration to 1, 2.5 and 5ug per well in next week and put them to scale-up in 10cm2 plate in presence of PURO. Cells reached to confluency with this amount of PURO, and then to confirm the successful KD, I performed WB of all the treatment concentrations.
Data showed the presence expression of AR protein. There is no change in AR expression as compare to control. However, cells are still surviving in this high Puromycin concentration.
I am not able to find the reason to this failure. Can someone suggest a troubleshoot?
Hi,
Is there any change in the expression of RNA? if there haven't any changes,maybe the shRNA sequences are not efficient,you should change other sequences.
Hi Gaurav,
It is possible the used prostate cancer cell line is resistant to Puromycin. A study published in 2013 found that Puromycin has inferior activity in comparison to other antibiotics selection-markers like Zeocin™.
For more information follow the link below:
https://www.ncbi.nlm.nih.gov/m/pubmed/23450727/
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