
shRNAs work in vitro, not in vivo
#1
Posted 27 July 2010 - 07:10 AM
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)
#2
Posted 27 July 2010 - 07:30 AM
"This is SPARTA!"
"I´m the goddamn batman"
#3
Posted 27 July 2010 - 07:32 AM
#4
Posted 30 July 2010 - 01:53 AM
#5
Posted 07 August 2010 - 12:24 PM
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.
- ravibiosciences and protienp63 like this
#6
Posted 09 August 2010 - 12:16 AM
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?
#7
Posted 10 August 2010 - 10:36 PM
#8
Posted 13 August 2010 - 06:25 AM
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?
#9
Posted 12 July 2011 - 10:53 AM
#10
Posted 27 July 2011 - 04:07 PM

#11
Posted 07 August 2011 - 03:20 AM
Please, does anyone know a shRNA inducible system that does not use Tetracycline ? Thank you