Figure 3.

RNA expression in CD4⁺ T cells sorted based on cell surface TCR expression. PCR of cDNA from single TCR transgenic mice and from sorted lymphocytes from doubly transgenic mice. (A) AND mice express Vα11 and Vβ3 mRNA, and MBP mice express Vα4 and Vβ8.2 mRNA, whereas T cells from AND/MBP mice sorted for cell surface expression of Vβ3 (AND) or Vβ8 (MBP) express mRNA from both transgenic TCR. (B) Both sorted Vβ3⁺ (AND) and Vβ8.2⁺ (MBP) T cells from double transgenic AND/D10 mice express mRNA from both TCR transgenes. Only Vα11 and Vβ3 cDNA are amplified from AND mice, and only Vα2 and Vβ8 cDNA from D10 mice are amplified. (C) All four TCR transcripts are amplified from D10/MBP T cells sorted as shown in Fig. 2D. Different Jβ usage in D10 and MBP allowed for each transcript to be separately amplified (as demonstrated in A and B), although both TCRs use the same Vβ8.2 gene segment. RNA was extracted and reverse transcribed, and the cDNA for the TCR α and β chains was amplified by PCR using primers specific for the Vα-Jα and Vβ-Jβ joins. The AND α chain was amplified using primers specific for Vα11 and Cα. Potential contamination of the cDNA samples with genomic DNA was evaluated by the use of primers specific for the second intron of the MHC class II Eb gene (42). Using identical conditions for PCR to those used to amplify the cDNA, no PCR product could be detected on ethidium bromide-stained agarose gels (data not shown).