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problems in getting stable clones


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#1 orna

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Posted 06 November 2004 - 09:47 PM

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. 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.

What do you think is the reason that the cells resistant to the antibiotic but do not express the siRNA?

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?

The reason I'm asking in this forum is because I don't think that I have problems with my siRNA.
Did you experience something like that?

Thanks a lot
Orna

#2 fred_33

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Posted 07 March 2005 - 12:42 AM

hi
in order to answer tothe fact siRNA cassette would be partially iontegrated in the genome, i don't think this is the best explanation. But if you think so, linearize your plasmid before transfection. you will ensure the complete integration.

Moreover, due to the fact hygromicin resistance gene and siRNA cassette are not under the same promoter, it is possible that you have a quite big difference of expression of this genes. But as you select cells that do express the HygroR gene, you get transfected cells. But you do not surely get siRNA expression. In order to get more results, you can make clones of raw cells and check of differences between several clones.

Finally, does your siRNA sequence have been published (and by the way is functionning) or is it just a try?

I'm sorry for late reply. I hope you have succeed since you posted this topic.

Fred




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