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Blood, Vol. 109, Issue 6, 2331-2338, March 15, 2007
Facilitating matched pairing and expression of TCR chains introduced into human T cells
Blood Kuball et al.
109: 2331
Supplemental materials for: Kuball et al, Vol 109, Issue 6, 2331-2338
Quantification of α and β TCR chains by real-time PCR
Expression of introduced  and  TCR chains was quantified by real-time PCR (TaqMan) using primers with specificity for the CDR3 region ( TCR chain: forward primer TTG TGC CCT CCC AGC CT, reverse primer TTT GGT CCC AGA TCC AAA GTA AA, probe 6FAM-AAG TCC CCG CTT GCT-MGBNFQ; TCR chain: forward primer TGT ATC TCT GTG CCA GCA GCT TA, reverse primer TCT GTG ACC GTG AGC CTG G, probe 6FAM-CCA GGC CTG TCC C-MGBNFQ; -actin primers and probes: TaqMan -actin detection reagents Hs99999903_m1; Applied Biosystems, Foster City, CA). RNA for v21 and v21 chains and for -actin was isolated from freshly transduced T cells and quantitated by RT-PCR by normalization to -actin copies. To assess the comparative levels of v21 RNA in T cells transduced with wt and Cys-TCR genes, transduced T cells were sorted for equivalent levels of v21 by flow cytometry, the RNA was isolated, and v21 chain message was quantitated by RT-PCR and normalized to v21 chain message. All sorts of TCR-transduced T cells yielded approximately 2-fold excess of the message compared with the -chain message for both wt chains (average for all sorts: 2.1 ± 0.6) and Cys-modified chains (1.7 ± 0.3).
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