I am studying a drug whose mechanism appears to be dependent on heat shock protein (HSP) function and I am attempting knockdown experiments to validate their independent implication.
Presently, I have had trouble with HSP72 knockdown in a glioblastoma cell line: controls with GAPDH siRNA using the same transfection protocol show >95% KD, however I can't get more than 5% KD of HSP72 using 3 different sequences targeted against the c-terminus (Ambion Silencer Select Pre-Designed series) using 100nM siRNA and measuring protein levels 48hrs following transfection (I am looking into transcript levels to see whether or not it is a protein stability issue).
In parallel, I'm considering HSP90 KD based on positive results with a pharmacological HSP90 inhibitor. Does anyone have experience with HSP90 KD? My initial worry about these experiments is that the level of cell viability will drop significantly given the dependence of cancer cell lines on HSP90 function.
Any comments, ideas, anecdotal experience is welcome and much appreciated. Thank you in advance.
Do you know for sure, that your HSP72 siRNA is working (e.g. from other publications)? If not, it is possible that your knock-down is bad, which can happen. Usually the companies send you free replacement siRNAs.
Regarding the HSP90, I wouldn't be surprised if it has an effect on cell viability, given its function and importance. But this is only guesswork.
http://www.nature.com/nchembio/journal/v3/n8/fig_tab/nchembio0807-455_F1.html
In the past I have done some HSP70 and HSP90 knockdowns and indeed could see a decrease in cell viability. This was especially apparent using lipofectamine as transfection reagent, which in itself could cause stress. I had much more success in keeping the cells alive using hiperfect and transfecting much less siRNA, reducing protein levels to about 20-30 %. This was sufficient for me to see the effects I was interested in.
Hello Sebastien,
I've done some Hsp90 knock down in the past and see a decrease in cell viability after 96h, but with good knock down efficiency (~90% after 48h). As transfection reagent I'm using HiPerFect but I change media after 6 h due to some toxic effects on my cells. Efficiency of my transfection is much more better when I'm using a reverse transfection protocol (Qiagen).
Hi Sebastien,
in my lab a lot of people worked on Hsp90 and they always told me that the KD caused a great reduction in cell number and viability, using siRNA from Dharmacon at 50 nM and using a 5 hours transfection with Oligofectamine. I do not know how much reliable is the KD, giving the harsh effects.
Bye,
Michele
Thank you very much for the responses. I really appreciate the information and suggestions.
Hi Sebastien,
Only a comment: you could be consider the effect of the HSP knockdown in other levels, like tissue or organ, the effect could be development disfunction. Thinking in applications.
Best Regards.
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