BSL8.4 TCR transgenic mice

BSL8.4 TCR transgenic mice (8.4a^+/−8.4b^+/−RAG1^−/−) expressing TCRα and TCRβ genes on the RAG1^−/− background that specifically recognize the Pb1 rodent malaria epitope were created. Briefly, the Vβ and Dβ segments of BSL8.4 were PCR-assembled with the Jβ2.1 genomic DNA segment (including 50 bp of J-C intron) and inserted into the XhoI/SacII sites of the pT_βcass cassette⁵⁵ (provided by the Benoist-Mathis laboratory, Harvard Medical School, USA). After removing the vector backbone by restriction digest, the gene cassette was microinjected into C57BL/6J oocytes. The resulting 8.4b^+/− line was bred to homozygosity on the RAG1^−/− background. To generate the 8.4a line, the full-length BSL8.4 TCRα cDNA sequence was inserted into the BamHI site of the pES4 vector⁵⁶ (provided by Prof. William Heath, University of Melbourne, Melbourne, Australia). The 8.4a^+/− line was crossed onto the RAG1^−/− background, but 8.4a^−/− mice were embryonic lethal. 8.4a^+/−8.4b^+/− RAG1^−/− mice were generated by crossing the resulting 8.4a^+/−RAG1^−/− and 8.4b^+/−RAG1^−/− lines; 98% of the CD8⁺T cells were positive for the SQLLNAKYL-H-2D^b (Pb1) tetramer²⁰ (Supplementary Fig. ⁴).