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RNAi in Jurkat cells


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#1 Chiel

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Posted 27 January 2005 - 03:44 AM

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

#2 fred_33

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Posted 31 January 2005 - 03:15 AM

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 (10g at least for a 15cm plate)
I filter the supernatant on 0.22m 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

#3 fred_33

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Posted 01 February 2005 - 04:27 AM

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]




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