FIG. 1.

Strategy for cloning and mutagenesis of MHV-68. Viral DNA and the linearized recombination plasmid containing the BAC vector sequences were cotransfected into eukaryotic cells to generate a recombinant virus. Circular DNA of the recombinant virus genome was isolated from cells and electroporated into E. coli. Mutagenesis of the MHV-68 BAC plasmid was performed with E. coli JC8679. The mutated BAC plasmid was retransformed into E. coli DH10B. In E. coli DH10B, the tetracycline resistance gene can be deleted by Flp-mediated recombination. The mutated BAC plasmid was transfected into eukaryotic cells to reconstitute recombinant virus. Propagation of the mutant virus in fibroblasts expressing recombinase Cre results in deletion of the BAC vector sequences. Circled arrows indicate FRT sites. P, loxP site; TR, terminal repeats.