RNA Interference - how to produce siRNA (Jan/27/2004 )
QUOTE (dpkp53 @ Feb 7 2004, 11:31 PM)
I am going to try the pSUPER retrovirus vector , which can stably express shRNA transcript in target cell line. have anybody tried this?
We do use this vector for shRNA study. Seem to work quite OK, at least so far. I have heard sometimes of the difficulty in maintaining stable cell line expressing shRNA, but the problem cannot be put due to the vector or other reasons. Because even when you use puromycin, there still are certain cells that naturally resistant. After a few generation, especially if your KD of the proteins cause some problems to cells, the uninfected cells will gradually kick the infected cells out of the pool. That is not to say in some case, the line between effective puromycin concentration (to kill all UNINFECTED cells) and killing concentratiion (to kill ALL cells) is WAY too thin. Very troublesome in these case. Depend on your luck then. But for just short term experiment, it should be OK. We use the vector for stable cell line and do success, too.
It is true though that one RNAi sequence may work well for some cell lines but not others. Have to test each time you want to use, and Dhamacon has suggested that we use pools (more than 1 sequence) for better results.