Gene Splicing by Overlap Extension or gene SOEing is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.

A model of demyelination induced by Theiler's murine encephalomyelitis virus (TMEV) was used to study differential regulation of class I MHC gene products in the brain and spinal cord of resistant (B10) and susceptible (B10.Q and B10.RBQ) mice. Allelic polymorphisms in the H-2D region, but not the H-2K region, play a primary role in determining susceptibility to late demyelinating disease. However, even though significant structural diversity distinguishes class I alleles, there are no discernible K or D-specific patterns of structural diversity within the peptide binding domains of these glycoproteins. Our hypothesis was that D region association of susceptibility to demyelination was related to differences in the expression of the K and D Ag in the central nervous system (CNS) after TMEV infection. Using allele-specific mAb and an immunoperoxidase technique, we demonstrated transient but equivalent increases in K and D Ag expression in the brain and spinal cord of resistant mice beginning 7 days after TMEV infection, which returned to baseline by 90 days. However, when genetically susceptible animals were examined, a significantly greater increase in D expression relative to K expression was seen in the brain and spinal cord at all post-infection observation periods. Immunosuppression of genetically resistant animals before TMEV infection, which results in viral persistence, was accompanied by equivalent increases in both the K and D Ag. Depletion of CD8+ T cells, but not CD4+ T cells, in susceptible mice ablated class I expression in the CNS in response to TMEV infection, implying that CD8+ cells contribute to the differential regulation of K and D Ag in the CNS. These findings are consistent with the hypothesis that differences in gene regulation may account for different roles of the K and D loci play in determining resistance and susceptibility to TMEV-induced demyelinating disease.