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stable clones - but expressing the gene! help!


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#1 orna

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Posted 31 October 2004 - 05:55 AM

Hi,
I had transfected my RAW cells with pRetroSuper-hygro that contain siRNA cassette.
After two weeks I got cells that survive the selection with hygromycin.
When I did a western blot I found that the cells expresses the protein that was supposed to be knockdown.

I know for sure that I got an efficient siRNA for my gene.
What do you think is the reason that the cells resistant to the antibiotic but do not express the siRNA?
Thanks a lot

#2 rassen

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Posted 31 October 2004 - 12:09 PM

I know for sure that I got an efficient siRNA for my gene.


Do you mean the designed siRNA has worked previously or you are sure your vector expresses the siRNA?

Is there any mRNA change in the transfected cells?
Is the western signal specific?
Are there any oligo design errors?

You can also check using Northern the expression of the hairpin RNA transcripted by your vector.

#3 orna

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Posted 31 October 2004 - 09:51 PM

It works in other cell lines. It works in reporter gene assays.
I didn't check the RNA levels and the western signals are specific.

Is their any chance that the vector partially integrated to the genome?
Or may be the gene I'm trying to silence is too important to survive that the cells manage to express the hygro resistance gene and not the siRNA?

thanks
Orna




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