Using pSuper retro - (Sep/12/2005 )

I’ve used siRNA to knock-down one gene: it worked fine, I got approx. 80 % knock down of the protein. I want to get a cell line constantly expressing shRNA, so I chose pSuper retro puro with the same sequence of hairpin as siRNA oligos. I did transient transfection to HeLa with a construct I’m not getting any knock down! The sequence inside of pSuper is fine according to sequencing. Transfection of pSuper is satisfactory, but I still expect to see some knock down in individual cells. Does anyone have any ideas where the problem might be?

Your question is a bit confusing: did you clone your shRNA into pSuper or into pRetroSuper (pSuperRetro)??

Your question is a bit confusing: did you clone your shRNA into pSuper or into pRetroSuper (pSuperRetro)??

Oh, I'm sorry, I've cloned shRNA into pSuper retro.

pRetroSuper contains the H1-RNA promoter, which is transcribed by polymerase III. When you have a TTTT or a AAAA within your sequence, this will lead to premature transcription termination.
Do you have such a sequence within your shRNA?

Hi Theo, withinh my sequence I just have TTT . Is it enough to get premature transcription termination?

I expect that TTT would be fine.

Did you already try to make retrovirusses from your pRS constructs to infect HeLa cells?
(You will need to make amphotropic virus or use HeLa cells which express the ecotropic receptor).

Theo,

No, I haven't made retrovirusses from my pRS constructs. We do not have HeLa cells which express the ecotropic receptor in our lab. We have never used any viruses in the lab, so we do not have resourses here. Do you think it is the only possibility to make it work?

The big advantage of using pRS and retrovirusses is that you will hit (nearly) 100% of the cells. If you are going to look at a knock-down of a protein, this is quit important, unless you can look at single-cell level.

Theo,
So, you say that it is not enought to do ''stable'' transfection, e.g. to transfect cells with pRS and then select single clones with puromycin?

Of course, I want to hit 100% of cells because I am not always will look at a single-cell level.

i want to good understand.
Do you select cells by puromycin or not?
If you cloned your shRNA into pSR, and not selected cells, i assume the shRNA expressed is a disadvantage for the cell and you slowely get only negative cells in your plate.
TTT is not a stop signal but it can lead to sporadic stops of transcription...

Infection of cells is a good way to hit quite all cells. But if you can't select the receiving cells it will be the same pb...
fred