I started off by creating a stable knockdown model using a control shRNA and two shRNA specific for my gene-of-interest. I achieved 90% knockdown after antibiotic selection, and the knockdown efficiency was tested routinely to make sure that it is stable.
I then use these cell lines for signalling studies, some results of which have already been shown with transient silencing of another cell line. However, this stable knockdown model was not able to reproduce the results seen with transient silencing, and whatever results I get from this model are barely reproducible. I took a step backward and did transient silencing using wild-type cells and was able to reproduce the results that were previously shown.
This may suggest that the shRNA model developed a compensatory/alternative mechanism,. But we are not sure how to explain this.
Any suggestions?
Hi Melissa. I always have issues with stable knockdowns. Plasmid or viral-based strategies for shRNA delivery lead normally to random integration in the cell's genome. This can cause positional effects (mutations in the already screwed-up genome of most cultured cell lines) as well as leading to different levels of shRNA expression depending on the area of integration. If you really want stable shRNA, I would suggest an inducible system where you can test the biological effect in the presence or absence of shRNA expression in the SAME cell line/population. My bottom line is that if I was to trust a result, I would have much more confidence with siRNA when compared to shRNA. This is from personal experience, I have been using siRNA with cultured cells from the early days, 2001...
Hi Melissa. I always have issues with stable knockdowns. Plasmid or viral-based strategies for shRNA delivery lead normally to random integration in the cell's genome. This can cause positional effects (mutations in the already screwed-up genome of most cultured cell lines) as well as leading to different levels of shRNA expression depending on the area of integration. If you really want stable shRNA, I would suggest an inducible system where you can test the biological effect in the presence or absence of shRNA expression in the SAME cell line/population. My bottom line is that if I was to trust a result, I would have much more confidence with siRNA when compared to shRNA. This is from personal experience, I have been using siRNA with cultured cells from the early days, 2001...
Melissa,
I agree with Miguel, we have used both transient knockdowns using electroporation of shRNA and lentivirus based stable shRNAs. Depending on your shRNA your cells might start to compensate by increasing or reducing the expression of other signaling proteins which will affect your signaling assays. It could also be that the protein being suppressed longterm may intrinsically control the genomic expression of other proteins. Since you have already used two different shRNAs the latter is more likely to be true.
Another question is do you also get 90% knockdown in your transient siRNA based transfections? knockdown efficiency which is likely higher in the stable cells may give you alternate results. Stables are always convenient and are my first choice, but if you are having problems you can try inserting your shRNA into lentiviruses carrying inducible shRNA promoters (pLKO.1) or use transient knockdown (electroporation of plasmid shRNA, or siRNA)
Hi Melissa, just to add something else for comments above. As Miguel said, for sure that insertion can induce problems. However, you did not mention how you make stable cell lines, whether you transfect shRNA plasmid and you do selection until you have plasmid integration into genome or you are infecting with retro/lenti viruses, because what will be inserted into genome could be different being much better by viruses (the insertion's problem is same for both, but by viruses you are sure that full cassette is inserted, plasmid integration could be only selection marker inserted without having shRNA). Since you are comparing always against scramble shRNA, if it is the case that insertion is causing troubles you should see insertion troubles with scramble too (sometimes it is better compare scramble against mock cells, should be same expression levels). Besides, you could do more than one infection in parallel to make sure have same results. A question, when you confirm KD, Are you performing WB? or by RT-qPCR?. Last comment, since siRNA normally is transfected in high concentration you could induce immune activation, toxicity, etc, on the other hand, shRNA produce siRNA but since it is made by cellular machinery its concentration is much lower, inducing less toxicity than transfected siRNA...this could be an issue if those effect are involved in the pathway that you are studying...Hope that it helps!
Like it was said before in the ShRNA assay the genome integration is random; thus, that's why you can have several different levels of suppression of your target protein.
Another problem of this assay is the off-target effects; however, it is easy to avoid this. You should use at least three constructs with different ShRNA sequences and a blast negative sequence (control). To affirm that in your assay there's no off-target effect you have to reproduce the same effects in all three constructs sequences.
I've worked with ShRNA and they can be tricky, but it is a very good tool if properly executed can give incredible results.
I hope it helps.
Hi Leonardo,
do you like to share your protocol which gave you good results for the shRNA transfection? I had also more success with siRNA transfection so far.
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