knocking down endogeous gene using shRNA - (Nov/28/2008 )

Hello everyone,

I have a problem of knocking down endogeous gene expression using shRNA constructs. I bouhgt those constructs from origene and they are supposed to be used in both transient transfection and retroviral infection. I tested those shRNAs in 293T cells by using a transient transfection with the co-transfection with my protein of interest, and the result turned out pretty good (3 out of 4 can successfully knock down the exgenous protein expression). However, those 3 failed to knock down the endogeous protein in LNCaP cells (an androgen independent prostate cancer cell line). I thought that maybe the problem of the infection at first, so i repeated the experiment again and got exactly the same result . Since those 3 shRNAs are supposed to target the coding sequence so i am really confused how come that happened. I called the company and they also had no idea what's going on. They said maybe the promoter driving the shRNA expression (U6 promoter which is activated by RNA PoII III) has been hypermethylated in LNCaP cells but i doubt that since i used very early passage cells after drug selection (2.0 ug/ml puromycin) for the western blot analysis, so from the point of kinetics that does not make sense. They also mentioned that maybe due to the processing of shRNA. But either of those two is hardly to test (ex, if i treat cells with demethylation agent, then it may lead to an overall change of gene expression).

Therefore, i would like to ask if you guys have ever experienced something like that or if you can give me some hinds about that. Thanks in advance.

Kun
eamil: zjhzlk2006@yahoo.com

A few questions/things to consider:

- You repeated the retroviral transduction, but did you have any control for good infection (a GFP marker or something, maybe?). Why don't you try the transient transfection on the LNCaP cells and see what happens?
- Is it possible that the siRNA binding site has been mutated? Maybe unlikely...

Hey miRNA man,

Thanks for your reply.

I had the GFP control for the infection and it worked pretty good. In case of doing LNCaP transient transfection, i have a little concern about doing that since the transfection efficiency is not that high (i had used lipofectamine 2000 and Fugene HD for some trial experiment and could only reach 30% cells transfected.), but i guess i may still give it a shot and that's what i'm doing right now.

It is also like what you said that the possibility of mutations at the binding sites maybe low, and i used 3 shRNA with different target regions and i hope i'm not that unlucky to get that tiny little chance with the mutations of all of them.

Still, thanks a lot

It looks like you've already got everything covered and controlled, I can't think of much else that could be going wrong. The only other possibility I can think of is maybe there is a mutation in Dicer or a RISC component so that even though the shRNA is transcribed, it cannot be processed correctly. Do you have a positive control, like gapdh shRNA that you can check?

In my experience, we have much much lower efficiency of knockdown when using virus compared to transient transfection. Using transient transfection in cell culture our knockdowns work beautifully: our positive controls are bright green, and western blots show virtually undetectable levels of target protein compared to mock-transfected cells.

However, using both adeno and lentivirus we've had nowhere near the same success, and have actually abandoned using this approach for one of our projects. I've spent a lot of time on the phone with Invitrogen and Applied Biosystems their explanation is that with knockdown copy number is everything.

DNA delivered to cells transiently is taken up in huge globs (e.g. with calcium phosphate, lipofectamine, etc.) that may have 20-40 copies of the shRNA DNA. Similarly, if you are transfecting siRNA directly to cells, very large numbers of the siRNA molecules are taken up. These approaches can very effectively knock down gene expression because the RNAi molecules are present in such high numbers.

In contrast, with viral infection you may have as little as one viral vector taken into a target cell. This means there will be only one copy of DNA present to direct synthesis of shRNA, as compared to upwards of 40 in a transient infection. This simply can't allow for the expression of enough shRNA to allow for efficient knockdown. However, one copy of a GFP reporter gene is sufficient to make cells green. This was our problem: cells infected with virus were green, but not showing knockdown. It sounds like you are having the same problem.

You can try to get around this by using a high MOI, but we found that at very high MOIs our cells died. We were simply never able to achieve satisfactory knockdown with virus, but have been lucky enough to since then receive a knockout.

Anyway. That's my experience. Good luck with your experiments.

Ginger

Hi Ginger Spice,

I appreciate for your suggestions.

I just had a committee meeting this morning and told my committee members about the current problems of shRNA knocking down. One of them told me they have also experienced something like that. The initail promoter driving the expression of their shRNA was also U6 promoter. And what they found was a perfect knocking down at the early passages and later the expression of their protein of interest bounced back after certain passages. So they switched to an expression vector using the H1 promoter and the result was amazing no matter how long you cultured the cells. He also suggest me to subclone my shRNA into that vector and then reassay my result. I hope it is also what happened in my case. You know sometimes there are too many possibilities and some are hard to test which totally drives me bananas and that is what science for