Viral Infection of Transgenic Mice Expressing a Viral Protein in Oligodendrocytes Leads to Chronic Central Nervous System Autoimmune Disease

Evans, Claire F.; Horwitz, Marc S.; Hobbs, Monte V.; Oldstone, Michael B.A.

DNA and RNA Analyses. Transgenic founders were identified by hybridization of NP-or GP-specific 32 P-labeled DNA probes (19) to slot-blots containing samples of DNA extracted from tail biopsies. Relative transgene copy numbers were determined by slot-blot hybridization as compared with known standards of gene copy number. Mice were characterized for RNA expression by RT-PCR on total RNA isolated as described (17) , using primers that hybridize within the transgene and a primer that hybridizes within PLP exon 7. RT-PCR products corresponding to correctly spliced NP transgenes were 510 bp (NP) and 780 bp (GP). RNase protection assays for cytokine mRNA expression were done on 2-4 mice using 15 g of purified total brain RNA or 1 g of poly-(A) Ï© mRNA as described (20, 21) . The relative amounts of cytokine RNA were quantitated by using the Series 400 PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Imager units (PI units) were normalized to equivalence based on values for rpL32 transcripts, which encode the L32 ribosomal protein (20, 22) .