Problem with shRNA stable transfection - (May/21/2006 )

Hi All....

I want to knockdown protein expression ...

So, I stably transfected jurkat T cell with my ShRNA inserted-plasmid that has puromycin resistance...
I grow them in 96 well then I transferred positive colonies to 24 well plate and so on....
However, when I pick the positive clones I didn't use cylinder ring, etc so I just transferred all the positive clones from each well in 96 well plate (each well in 96 well plate after puromycin selection resulted in 2 or 3 clones colonies).

After several weeks, I want to confirm the knockdown by western blot...
and I got a weird result...:
Western blot form a first lysate showed that I got a little bit knockdown. I want to confirm this by did the second western blot using a new lysate and the result is that the protein expression is completely dissapeared and (Ialready check the protein loading control too) so this is a good thing but I still confused how come the intensity of knock down is different when I do a western blot using a different lysate ...So, I did the third western blot also with a new lysate and the result is I only got very little knockdown....

I am so confusing at this point ....

Is anyone has an advice or maybe know why this could happened?

Thank you in advance

Hi,

I stably transfected NB4 cells with shrna and I too get similar results, the wb's are often showing different knock down effects. Perhaps it depends on the growth phase of the cells, I find if my cells are confluent the protein levels are higher. Are you still treating with puromycin every 3 days?
How did you transfect the jurkat? I used electroporation to transfect my NB4 cells.

Hi,

I'm also having trouble with getting a stable shRNA transfection.

I have tried transfecting shRNA individual contructs into NB4 cells by electroporation in 6-well plates. After 48 hours of transfection, cells are centrifuged and new medium's added together with selection drug Purmomycin (1ug/ml). Medium is changed after every 48hours subsequently.

However, it seems like my cells are all dead.

Hope to hear some opinions/suggestions. Thanks!

hannah

I am using lentivirus to infect my cells to transfer shRNA. The infect cells nicely.

Knocking down the protein depends on many factors.

I get different levels of knockdown depending on how long the cells r tranfected with the shRNA. Also on the amount of total protein being loaded in WB.

If u repeatedly get a low knockdown, then its probably true.