The SIN-(NFAT)_x-GFP retroviral construct.

The SIN-(NFAT)_x-GFP–retroviral vector contains a puromycin resistance gene for selection purposes, and EBNA sequences to maintain high copy numbers of the transfected construct in the amphotropic producer line Phoenix, enabling the production of high titer viral supernatants. Transduction of target T cells with the retrovirus, containing the 5′LTR-(NFAT)_x-GFP-3′LTR only, ensures that expression of the reporter gene is dependent on binding of transcription factors to the multiple NFAT-binding sites, because an introduced deletion in the U3 region of the 3′LTR (which, on integration, will function as the upstream LTR) prevents promoter activity of this LTR. Thus only activation of the T cell will lead to expression of GFP. eGFP, enhanced EFP.