RNAi in Jurkat cells - (Jan/27/2005 )
I have difficulties establishing RNAi in Jurkat cells (non-adherent cells) with constructs that do work in several adherent cell-lines. I am using shRNA in the retroviral pSUPER vector. Does anyone experience the same problem and does anyone have an idea of how to solve it ?
from my knowledge pSUPER is not a retroviral vector...
I used last year pFHM IRES NEO and passed to pSUPER due to the fact it contains the puromycine resistance gene (instead of neomycine resistance gene for pFHM IRES Neo).
Now i use the pLN vector. This one is retroviral.
293 Pack cells are transfected with this vector (10µg at least for a 15cm plate)
I filter the supernatant on 0.22µm filters and the add sequabrene (or polybrene) and allow the infection for 24h minimum. for complete protocol see :
see also the oligoengine page at http://www.oligoengine.com/pSUPER_New/pSUPER_Main2.html
they point out the fact that the retroviral alternative of pSUPER vectors is pSUPER retro
I too am having similar problems in getting knockdown in Jurkat cells with shRNA constructs that are working in overexpression studies in 293 cells. I have also tried transfecting siRNA versions but these also seem to be inefficient at knocking down the endogenous protein. I have just been using standard electorporation to transfect the cells I am getting good transfection efficiency (40% with shRNA and >90% with the siRNA) but no knockdown. I was just wondering if you had solved your problem and have any tips for me? Any suggestions would be much appreciated