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. 2012 Dec 18;1(12):e63. doi: 10.1038/mtna.2012.52

Figure 3.

Figure 3

The order of TCRα and β genes connected by the T2A peptide affects TCR cell surface expression. (a) Schema of the retroviral vectors used to transduce the PBMCs. The pMS3-a2Ab, pMS3-b2Aa, pSplice-a2Ab-siTCR, and pSplice-b2Aa-siTCR were constructed to express WT1-, MAGE-A4-, hTERT-specific TCRs. (b,c) PBMCs from more than three different donors were transduced with serially diluted retroviral vectors and used for tetramer staining and proviral copy number analysis. (b) Representative flow cytometry analysis of PBMCs transduced with WT1-, MAGE-A4-, and hTERT-specific TCR-expressing vectors, with comparable proviral copy numbers indicated in parentheses. The MFIs of the tetramer are also indicated. (c) The percentages of tetramer-positive cells among the CD8+ cells in MS3-a2Ab- (open circles), MS3-b2Aa- (closed circles), Splice-a2Ab-siTCR- (open triangles), and Splice-b2Aa-siTCR- (closed triangles) transduced PBMCs are plotted according to the copy number. The percentages of tetramer-positive cells per proviral copy were calculated and evaluated by Student's t-test. *P < 0.05. The PBMCs used in b and c are from separate donors. Ψ, packaging signal; hTERT, human telomerase reverse transcriptase; LTR, long terminal repeat of M-MLV (5′LTR) and MSCV (3′LTR); MFI, mean fluorescence intensity; MLV, murine leukemia virus; MSCV, murine stem cell virus; PBMC, peripheral blood mononuclear cell; PGK, phosphoglycerate kinase promoter; SA, splice acceptor; SD, splice donor; siRNA, small-interfering RNA; T2A, -SGSG-linker peptide+T2A peptide; TCRα, codon-optimized TCRα chain; TCRβ, codon-optimized TCRβ chain.