Expression of Bcl-xL and Survivin using a 2A gene sequence in primary CD8+ T cells. (a) Schematic representation of the retrovirus construct expressing Bcl-xL and Survivin. Ψ, packaging signal. 2A, picornavirus self-cleaving 2A sequence. (b) Genes of Bcl-xL and Survivin were linked with T2A sequence and were subcloned into Mig vector (Mig-bcl-xL-2A-survivin), which was confirmed by digestion with restriction enzymes, showing fragments of Survivin (~500 bp), Bcl-xL-2A (~750 bp), and bcl-xL-2A-survivin (~1,200 bp). (c) Naive CD8+ T cells from OT-I TCR transgenic mice were stimulated with peptide/APCs. On day 2/3, T cells were transduced with retroviral vectors expressing GFP (Mig), GFP with Bcl-xL (Mig-bcl-xL), GFP with Survivin (Mig-survivin), or GFP with Bcl-xL and Survivin (Mig-bcl-xL-2A-survivin). On day 5 of primary culture, GFP+ CD8+ T cells were sorted, and protein expression of Bcl-xL, Survivin, and β-actin was determined by western blotting. Data are representative of three independent experiments.