2 replies to this topic

[Chiel]

Hello everyone,

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 ?

Thanks!

Chiel

[fred_33]

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 :
http://www.stanford....helper_dep.html

good luck

[fred_33]

see also the oligoengine page at http://www.oligoengi...UPER_Main2.html

they point out the fact that the retroviral alternative of pSUPER vectors is pSUPER [B]retro[B]