Stable Transfection with siRNA and RNAi

[kiki06]

As you know, there are many new vectors which allow the cloning of RNAi expression cassettes.

These can be transfected into cells, and even stable cell lines can be created by selecting methods such as Neomycin.

Does anyone have a general protocol for the stable transfection of RNAi into any cell type?

thanks in advance
Kiki

[jpap]

I have successfully used several constructs for stable knockdown in cancer cells. It would be useful, if possible, to try more than one promoter and targeting sequences and find which one works best in your system.
As for the delivery methods, I have tried "conventional" lipid-based transfection methods (such as Lipofectamine) and retroviral infections. I did not modify the standard protocol at all, I did the plating, transfection and drug selection as I would do in a stable overexpression experiment. I hope that helps a bit.

[babu]

I am having a KB stable cell line with tetR gene. I want to stably transfect a siRNA vector having U6 promoter...is it possible..if so how?pls give your valuable suggestions. thnx

[Luciferase]

pSUPER....it works. but any vectors with the proper promoter fitting your construct will do.