A problem about infecting primary T cells - (Mar/31/2008 )

In this experiment, I am trying to over-express a gene in primary T cells using the Invitrogen 4 plasmids system "pRRL-expression plasmid/PLP1/PLP2/PLP-VSVG". Here is my protocol:


Primary T cells were activated with plate bound anti-CD3/CD28 mAb (2ug/ml) for 24 hours before lentiviral-infection of pRRL-eGFP and pRRL-gene-IRES-eGFP. 6 days later, a FACS assay was perfomed to determine the infection efficiency. I noticed the infection efficiency dropped significantly in the pRRL-gene-IRES-eGFP. A representative result was shown in this link.

http://www.protocol-online.org/forums/inde...showtopic=35362


Could you give me some advice? How about adding a promoter after IRES? I mean “pRRL-gene-IRES-promoter-eGFP”? Or, change another stronger promoter?


Thank you very much!

I guess you could try with 2 promoters for your gene and GFP. Is the IRES part of the commercial vector or have you added it and are you sure it works? This should work to give you expression of your protein and the GFP. Can you look for expression of your gene/GFP to see if the gene is expressed by the IRES isn't working? Could your gene of interest be toxic to the cells?

Hope that helps,
Ceri