Critical Role of Perforin-dependent CD8+ T Cell Immunity for Rapid Protective Vaccination in a Murine Model for Human Smallpox

Kremer, Melanie; Suezer, Yasemin; Volz, Asisa; Frenz, Theresa; Majzoub, Monir; Hanschmann, Kay-Martin; Lehmann, Michael H.; Kalinke, Ulrich; Sutter, Gerd

RNA isolation and amplification of human GAPDH cDNA were performed as described [95] using 26 amplification cycles. Similarly, amplification of murine GAPDH cDNA was performed with the sense primer 59-GAC AAC TCA CTC AAG ATT GTC AG-39 and the antisense primer 59-GTA GCC GTA TTC ATT GTC ATA CC-39, resulting in a product size of 540 bp. Amplification of VACV B8R gene (GenBank accession no. AY603355) was undertaken using the sense primer 59-TAA AAA TTA TGG CAT CAA GAC G-39 and the antisense primer 59-ACA TCT TCT TTG GAT CTA ATT GC-39, resulting in a product size of 495 bp for MVA, and 540 bp for VACV strain Elstree. The MVA E3L gene orthologue has been amplified using the sense primer 59-TTA CTA GGC CCC ACT GAT TC-39 and the antisense primer 59-GTT CTG ACG CAG AGA TTG TG-39, resulting in a product size of 406 bp. Primer pairs were designed using Primer3 software [96] . All oligonucleotides were synthesized by Eurofins MWG Operon GmbH (Ebersberg, Germany). PCR products were run on a 1.5% agarose gel and stained with GelRed purchased from MoBiTec (Göttingen, Germany). Gel pictures acquired by a CCD camera were analyzed using the Photo-Capt 12.4 software (Vilber Lourmat, Eberhardzell, Germany).