4 replies to this topic

[litos]

Hello everyone,

I am experiencing trouble in screening for an effective shRNA sequence. There are varios approaches (western/northern blot, flow cytometry...). As RNA interference does not produce an absolute phenotype (ie total absence of the target protein), I think the best approach for an immediate sequence selection is flow cytometry. I have designed 4 shRNA candidate sequences which are currently cloned into an expression vector, also expressing the eGFP. My target protein is cloned into an interim expression vector which does not produces any reporter tag or protein. I tested two out of four sequences by cotransfection in 293 cells and subsequent immunohistochemistry (72h after cotransfection) but none of them produced any clear effect. Nonetheless, I performed cell counts to be sure that my shRNAs were not working at all. I couldnt find any drop in the number of cells expressing my target protein, but I found what seemed to be a slight drop in the intensity of fluorescence.

I think the best course of action now, before I test the last two shRNAs sequences, is to subclone my target protein into an expression vector that produces a fluorescent reporter protein different from eGFP. The target protein must be cloned in-frame with the reporter protein or the latter be expressed from the same messenger via an IRES signal. Then, I should perform a cotransfection assay and analyze the fluorescence levels by flow cytometry. I think that is the faster and most precisse way to compare the fluorescence between different conditions. ¿Do you think I am right? Any advice is welcomed...

Finally, I have a more prosaic and methodology-related question. I have been reading some protocols to test shRNAs by cotransfection. Some of them propose to mix the target cDNA plasmid with the plasmid containing the silencing cassette in 1:5 molar ratio (in my experiment, I used an 1:1 ratio), but they dont explain this point further. ¿Can anyway shed light on this issue?

Thanks in advance.

Edited by litos, 01 July 2009 - 10:55 AM.

[Functional Screens]

Why not use Western Blot instead of FACS.

I will start from target:shRNA 1:10 for co-transfection to validate the effective shRNA.  To screen the potent shRNA, I will change the ratio to 1:5 or 1:3.  The process of shRNA and some other endogenous interferences might not make the shRNA to sufficiently suppress the gene expressed from the vector with the equal molar ratio to shRNA vector.

[litos]

Thank you, Functional    That sounds fine, but I still have some doubts. My objetive screening different shRNAs is to select one for subsequent production of viral particles in a packaging cell line. Then, I would want to perform a co-infection experiment in-vitro, to validate the sequence in my target cell type (neural stem cells). Finally, I would try that infection in vivo.

Therefore, ¿where could I put the threshold in my cotransfection experiments to give the OK to one shRNA sequence? In other words, if I try, lets say, a 1:10 ratio in my cotransfection experiment and see good interference, ¿should that sequence work in my co-infection experiments? I know that is a tricky question, but I am a bit lost...

Thanks a lot.

Edited by litos, 07 July 2009 - 01:54 AM.

[litos]

Thank you, Functional    That sounds fine, but I still have some doubts. My objetive screening different shRNAs is to select one for subsequent production of viral particles in a packaging cell line. Then, I would want to perform a co-infection experiment in vitro, to validate the sequence in my target cell type (neural stem cells). Finally, I would try that infection in vivo.

Therefore, ¿where could I put the threshold in my cotransfection experiments to give the OK to one shRNA sequence? In other words, if I try, lets say, a 1:10 ratio in my co-transfection experiment and see good interference, ¿should that sequence work in my co-infection experiments? I know that is a tricky question, but I am a bit lost...

Thanks a lot.

Edited by litos, 07 July 2009 - 01:56 AM.

[Functional Screens]

Well, if you can get 2 out 5-10 shRNAs giving you more than 70% knockdown, then you have to use them and target:shRNA ratio does not matter.
If you have more than 2 shRNAs are effective, then I will try to change the ratio till I have the final TWO standing out.  Those two should be the most potent shRNAs which I will use for my experiments.  So, I will say the threshold is "can you get two potent shRNAs showing more than 70% knockdown"....................

Edited by Functional Screens, 07 July 2009 - 03:50 PM.