question about shRNA knocking down

[zjhzlk]

Hello everyone,

I have a problem of knocking down endogeous gene expression using shRNA constructs. I bouhgt those constructs from origene and they are supposed to be used in both transient transfection and retroviral infection. I tested those shRNAs in 293T cells by using a transient transfection with the co-transfection with my protein of interest, and the result turned out pretty good (3 out of 4 can successfully knock down the exgenous protein expression). However, those 3 failed to knock down the endogeous protein in LNCaP cells (an androgen dependent prostate cancer cell line). I thought that maybe the problem of the infection at first, so i repeated the experiment again and got exactly the same result . Since those 3 shRNAs are supposed to target the coding sequence so i am really confused how come that happened. I called the company and they also had no idea what's going on. They said maybe the promoter driving the shRNA expression (U6 promoter which is activated by RNA PoII III) has been hypermethylated in LNCaP cells but i doubt that since i used very early passage cells after drug selection (2.0 ug/ml puromycin) for the western blot analysis, so from the point of kinetics that does not make sense. They also mentioned that maybe due to the processing of shRNA. But either of those two is hardly to test (ex, if i treat cells with demethylation agent, then it may lead to an overall change of gene expression).

Therefore, i would like to ask if you guys have ever experienced something like that or if you can give me some hinds about that. Thanks in advance.

Kun
eamil: zjhzlk2006@yahoo.com

[jonmoulton]

Doing your transient knockdowns with an oligo instead of a construct avoids some of the potential problems. You could express your shRNA in a cell-free system then transfect the cells. This at least would eliminate some of the possible problems and you can determine whether the shRNA sequence is failing or its expression in the cells is the source of the problem.