Figure 1

An in vivo strategy for functionally searching the parapoxvirus ovis genome for useful ORFs. The genomic sequence of parapoxvirus ovis (ORFV) strain D1701 was determined, and ORFs were identified. Sequence results were used to design PCR primers for gene amplification. The library of parapoxvirus ORFs (ORFeome) was generated and then partitioned into 27 pools of 8 to 10 ORFs each. Mammalian promoter and terminator expression elements were added to each pool, treated to expose single stranded end sequences, and then assembled into LEEs by sequence specific annealing. The ORF-expressing LEE pools were precipitated onto gold microparticles and biolistically delivered into the abdomen skin of BALB/c mice. To limit the number of animals used, each mouse was gene-gun inoculated with four to six different LEE pools at distinct abdominal sites. Each pool was tested in triplicate, in 3 different mice. Tissue sections were harvested 4 days later, cryo-sliced and immuno-probed to identify MHC II expression (I-A^d/I-E^d) and visualize cell morphology.