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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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pSuper
Posted by: agne (IP Hidden, New member, 1)
Date: September 12, 2005 05:45AM
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?
Re: pSuper
Posted by: 3'-UTR (IP Hidden, Unregistered user, )
Date: September 14, 2005 01:42PM
Be sure to rule out IFN effect on your "successful" siRNA knockdown at very beginning.
Be awared that transient siRNA effect sometimes is difficult to translate into stable shRNA expression due to unknown reasons, could be the cell type-specific DICER activity/amount/efficiency, siRNA molecule # per cell, target sequence, off-target effect......
Re: pSuper
Posted by: jonmoulton (IP Hidden, Junior member, 15)
Date: September 14, 2005 02:13PM
You can confirm the first siRNA knockdown using Morpholino antisense, which does not induce TLR-mediated innate immune responses such as the interferon system. Unfortunately Morpholinos are only useful in transient transfection, they cannot be expressed in cells.
- Jon Gene Tools, LLC
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