Problems with gene knockdown - No knockdown with validated shRNAs :( (Aug/19/2009 )
Hi
I am using shRNAs to knockdown target genes. But I have been having trouble observing the knockdown. I have 2 projects going on. shRNAs are validated by Luciferase Assay. In one project, the selected shRNA failed to show any reduction in protein expression levels in WB. I then infected the cell line with 5 different shRNA constructs and still none of them showed any reduction. Of the 5, 3 showed good efficiency in luciferase assay. In the other project, we selected an shRNA from the assay and generated a mouse using lentiviral transgenesis. The construct has the marker protein and the shRNA under separate promoters. The mouse expresses the marker protein in almost all cells abut can still produce loads of the target protein. 
I am really confused and would appreciate some help. Could anyone please give me some advice? Thanks
Julz
Hi there!
From my experience, the luciferase assays do not necessarily reflect the in vitro or in vivo knockdown...which luciferase system are you using? in systems like psicheck2 (promega) where fusion mRNA are generated, the secondary structure and therefore the target sequence accesibility seems to be different from the "normal " secondary structure of the target mRNA (has been published as well, can't find the paper at the moment...). This can change the outcome. I had many constructs which looked amazing in the Luc assay (85 - 90 % knockdown) but did not have any effect in cells naturally expressing the mRNA.
Maybe there are more people here with the same experience?
Stardust
Interesting....2 questions - first, for your luciferase validation, is the shRNA promoter the same as used in your transgenic mice? And secondly, you say you have 2 promoters for the shRNA and reporter, but are they the same promoter (i.e. both PGK etc)?
I also wonder if you can design an assay (PCR?) to directly test for the presence of the shRNA? Finally, the half life of luciferase protein could be a lot shorter than your GOI, maybe you could look at later time points after introducing the shRNA (at least maybe possible in the cells, maybe not in the mice unless it's a conditional system)??
My two cents,
1. feedback regulation: you were able to knowdown gene in reporter assay but the endogenous gene (for example, some metabolic genes) might not be able to knockdown due to feedback regulation.
2. gene is required for RNAi or affecting the RNAi efficacy while been knocking down
3. gene family/homologue: you can see good knowdown in reporter assay, but the antibody you used for Western is blotting the whole family
4. promoter silencing: you are using polII promoter (especially CMV) instead of polIII promoter to express shRNA, the polII promoter got silenced in transgenic mice.
5. higher titer might help but no guarantee
6. cell sorting to collect the top 10-20% of infected cells if you can use GFP or RFP
stardust on Aug 19 2009, 12:13 PM said:
From my experience, the luciferase assays do not necessarily reflect the in vitro or in vivo knockdown...which luciferase system are you using? in systems like psicheck2 (promega) where fusion mRNA are generated, the secondary structure and therefore the target sequence accesibility seems to be different from the "normal " secondary structure of the target mRNA (has been published as well, can't find the paper at the moment...). This can change the outcome. I had many constructs which looked amazing in the Luc assay (85 - 90 % knockdown) but did not have any effect in cells naturally expressing the mRNA.
Maybe there are more people here with the same experience?
Stardust
Hello
Thanx for the reply. I am using psicheck2 from promega. From your reply it seems the system is not that reliable. Is there any other way that you can suggest for me to test the knockdown efficiencies?
miRNA man on Aug 19 2009, 05:25 PM said:
I also wonder if you can design an assay (PCR?) to directly test for the presence of the shRNA? Finally, the half life of luciferase protein could be a lot shorter than your GOI, maybe you could look at later time points after introducing the shRNA (at least maybe possible in the cells, maybe not in the mice unless it's a conditional system)??
Hi
Thanx for the reply. To answer your questions, I used the same construct for both luciferase validation and for generating the mice. I have the reporter under ubiquitin promoter and the shRNA under U6 promoter. So it is possible that one is on and the other is off. I could probably do a northern blot to test the presence of shRNA. And for the project with the cell lines, the vector has both the reporter (antibiotic resistance) and the shRNA under the same promoter and so as long as the cells are resistant to antibiotic, should express the shRNA. I usually test the cells by Western blot only after weeks of growing them in culture. I tested the antibody specificity using a blocking peptide and it worked fine. All the samples have similar band intensities. The protein is expected to be expressed in low quantities in the cell line.
Functional Screens on Aug 19 2009, 10:34 PM said:
1. feedback regulation: you were able to knowdown gene in reporter assay but the endogenous gene (for example, some metabolic genes) might not be able to knockdown due to feedback regulation.
2. gene is required for RNAi or affecting the RNAi efficacy while been knocking down
3. gene family/homologue: you can see good knowdown in reporter assay, but the antibody you used for Western is blotting the whole family
4. promoter silencing: you are using polII promoter (especially CMV) instead of polIII promoter to express shRNA, the polII promoter got silenced in transgenic mice.
5. higher titer might help but no guarantee
6. cell sorting to collect the top 10-20% of infected cells if you can use GFP or RFP
Hi
Thanx for the comments. Since I have problems with 2 separate projects, I am not sure whether it could be feedback regulation or interference with RNAi. The western blot is done on cells which have been infected with the virus containing the shRNA construct. The construct has both shRNA and antibiotic resistance under the same promoter and so all the cells growing in the media should have the knockdown. I lyse the cells only after growing them in culture for weeks and the antibody is specific and published in literature. The virus I used to generate the mice usually have high titres.