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.

Generation of Transgenic Mice. The LCMV Armstrong NP cDNA was prepared by BamHI digestion from plasmid pARMNP, which contains the entire coding sequence of the NP. The full-length cDNA of the LCMV Armstrong GP was obtained by RT-PCR amplification of viral RNA. Total RNA was purified from the brain of a mouse that had been persistently infected with LCMV Armstrong since birth. cDNA was made using random hexamer primers and murine Maloney leukemia virus reverse transcriptase as described (17) . PCR amplification of the viral GP was done using primers that hybridize to the 5 Ј and 3 Ј ends of the GP sequence: primer 1, which hybridizes to bp 1-26, 5 Ј -CCGGGGATC-CTAGGCTTTTTGGATTG-3 Ј ; and primer 2, which hybridizes to bp 1553-1585, 5 Ј -GGGGATCCTGTTCTTCAGCGT-CTTTTCCAGAC-3 Ј . PCR was done using Taq Polymerase (Perkin Elmer Cetus, Norwalk, CT) following reaction conditions indicated by the manufacturer and performing 35 cycles at 95 Њ C for 1 min, 60 Њ C for 1 min, 72 Њ C for 3 min. The 1575-bp product was cloned into the TA vector system (Invitrogen, San Diego, CA) and then digested with BamHI. The NP and GP cDNAs were cloned into the BamHI site of pMBP001, which contains sequences from Ϫ 1907 to ϩ 36 of the MBP promoter, followed by a polylinker region linked to a portion of the proteolipid protein (PLP) gene as described (18) . The PLP sequences provided splice and polyadenylation signals and included PLP exon 6, intron 6, and exon 7. Linear DNA fragments containing the regulatory regions plus NP or GP sequences were obtained by NotI digestion. The DNA was microinjected in The Scripps Research Institute Transgenic Facility into BALB/cByJ ϫ C57BL/6J fertilized eggs. LCMV-NP transgenic mice were bred to BALB/ cByJ (H-2 d ) mice, and the LCMV-GP transgenic mice were bred to C57BL/6J (H-2 b ) mice. MBP-␤ -galactosidase ( ␤ gal) transgenic mice (18) were bred to BALB/cByJ and C57BL/6J mice. All nontransgenic breeder mice were obtained from the breeding colony at The Scripps Research Institute (La Jolla, CA).