siRNA Stable transfection in human fibroblast cells - (Oct/17/2005 )

Hi, guys,

I have a really difficult time to find a stable celll clone that the targe protein (a nuclear protein) is dramatically down-regulated by siRNA. I have 6 siRNA, tested them by transient transfection on HEK 293 cells, one of them worked on HEK293 cells. So I constructed a siRNA (pSilencer3.1) vetor using this siRNA. No luck after screening 100 cell clones. It's not pratical to directly test siRNA on the paticular cell strain I am working with, this cell strain can only used for stable transfection by picking clones. I plan to test more siRNA and try retrovirus system for stable transfection. Any suggestions and ideas?

Thanks.

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Hello,

Have you tested your shRNA construct in other cell lines using transient transfection? Sometimes an siRNA works but shRNA expressing it may not work. So before going straight for stable transfection, try the shRNA in transient transfection.

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hi
pcrman is right. expression of an siRNA can be reduced/enhanced by the loop sequence you use.
i've you monitored your transfection efficiency?

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I tried transient transfection in 293 cells using the shRNA construct, it worked. So I used it for stable transfection which turned out didn't work. I am worrying about how stable shRNA is, e.g, it worked for couple of days, and somehow it stopped working although the cells are still resistant to the selection drug. When I ordered siRNA vector from siRNA, such as 3.1 and 4.1 version, they all claimed"stable up to 3 or 6 monthes", what's if more than one year?

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hi
t think that stable for up to 3 to 6 month means that you need at that time to retransfect cells.
Do you have any knock down? Can you detect your siRNA?

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Possible that complete ablation of your protein is cell lethal.
Some people use vectors with GFP to moniter their infectivity.

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293HEK were never fibroblasts, unless you performed a Hwang-stile miracle

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