Figure 7

Ectopic expression of IL2RA and IL15IRA. (A) The murine cDNAs for IL2RA and IL15RA were cloned into MP91-EGFP upstream of the IRES. IRES, internal ribosomal entry site; wPRE, Woodchuck hepatitis virus posttranscriptional regulatory element. To detect cells transduced with different vectors fluorescent marker Venus substituted for EGFP in MP91-IL2RA and Cerulean in MP91-IL15RA (B) Primary mature T cells were transduced either with LMO2, IL2RA or IL15RA alone or in combination: LMO2 + IL2RA, LMO2 + IL15RA, IL2RA + IL15RA and LMO2 + IL2RA + IL15RA. As control populations, control vector MP91-EGFP-transduced and nonmodified T lymphocyte cultures were initiated. During follow-up culture, marker gene expression served as the level of gene modification. Gene marking increased solely in IL2RA- (6% to 100%) and LMO2+ IL2RA-cultures (3% to 100%). (C) Gene modified T cells were kept under standard cell culture conditions for several weeks and cell counts determined regularly. MP91-EGFP and nonmodified T lymphocytes served as control populations. Only the combination of IL2RA with LMO2 supported long-term growth of mature T lymphocytes. Relative cell counts over time are depicted.