10 replies to this topic

[ambv34]

I am facing an annoying problem, and I would be more than happy if somebody has some ideas regarding what might be happening here (sorry that my message will be a bit long).

I made shRNAs against two targets, and selected the two best ones for each target based on in vitro knock-down on luciferase reporters + endogenous mRNA.

Then I injected some mice...
- same cassette in the plasmid used in vitro and the viral vector used in vivo
- 100% transduction of the target cells (hepatocytes)
- reporter gene is expressed and doesn't get silenced
- siRNAs are expressed
- there's even a slight liver toxicity in a few mice
... and yet, there's no knock-down, with none of my four shRNAs.

To make it easier, some colleagues of mine did pretty much the same with one single shRNA after screening only with luciferase reporter - same virus, same dose, same protocol, same control, etc... - basically only the target is different, and it worked like a charm.

Oh, and of course it's unlikely that it's because of my targets, because at least one of them has been knocked-down previously in vivo by another group. And one of my four shRNAs is the one this other group published.

I didn't manage to make up an explanation that would make sense, so far... Anybody?? (pleeaase)

[laurequillo]

how did you check the expression of your target gene? WB, RT-PCR? Sometimes you cannot see the silencing at protein level, but it is clear at RNA level.

[ambv34]

No, qPCR, using the same primers as the ones used for in vitro. And the cell line used is derived from the mouse genotype I used.

[ambv34]

It seems that nobody had this problem before...?

[Functional Screens]

1. One of your target gene has feedback regulation
2. The primer sets you used for genome-typing are not good for RT-qPCR or qRT-PCR especially when you have genomic DNA contamination.
3. Dosage issue or shRNA potency; the same dosage works for your colleague because his target gene can be silenced in a lower concentration of shRNA.  You might want to screen more potent shRNA by playing around the ratio of shRNA vector/luc-target vector (e.g. 1:1 or 1:5).  Then use the shRNA for your in vivo experiments.

[ambv34]

Functional Screens, on 07 August 2010 - 12:24 PM, said:

1. One of your target gene has feedback regulation
2. The primer sets you used for genome-typing are not good for RT-qPCR or qRT-PCR especially when you have genomic DNA contamination.
3. Dosage issue or shRNA potency; the same dosage works for your colleague because his target gene can be silenced in a lower concentration of shRNA.  You might want to screen more potent shRNA by playing around the ratio of shRNA vector/luc-target vector (e.g. 1:1 or 1:5).  Then use the shRNA for your in vivo experiments.

Thanks for your reply!
Feedback regulation is I guess a possibility at least for one target... even if the same happens with the other target (already published).
About primers, I've tested a few sets already, all giving the same results, and my samples are all DNase-treated, and my RT- controls are negative.
For my luciferase screen, I have indeed used several ratios, 1:20, 1:4, 1:1, 1:0.2, with the idea to pick the more potent shRNAs, but they all showed the same trend... Would you expect that this has an influence?

[Functional Screens]

I will definitely pick the shRNA always gave more than 70% knockdown in your 1:1 or 1:0.2 reporter assay.  You might want to introduce your luciferase reporter in vivo for confirmation.

[ambv34]

Functional Screens, on 10 August 2010 - 10:36 PM, said:

I will definitely pick the shRNA always gave more than 70% knockdown in your 1:1 or 1:0.2 reporter assay.  You might want to introduce your luciferase reporter in vivo for confirmation.

Well, most of my shRNAs give more or less 50% knockdown at these ratios, and at higher ratios the % of knockdown is increased (70-80%) but still pretty similar between different shRNAs. So luciferase didn't really help me in my choice. I then decided to pick the best ones based on endogenous knockdown in vitro (70-80%).
I thought that endogenous knockdown in vitro would be more predictive of the in vivo outcome compared to luciferase reporters... Do you disagree?

[Xavier2]

Have you tried synthetic siRNA instead of ShRNA, they are more potent and easier to design than shRNA/

[DNA_modified_cabbage]

I've been testing same for couple of month now and faced this problem twice. Solution is obvious - since I've tried to use hominidae species it improved significantly! It's cause reaction on syntheses produce more efficient ways of signals sent by hepatocytes cells...

[canine]

Hi there,

Please, does anyone know a shRNA inducible system that does not use Tetracycline ? Thank you