Generation of mutant murine cytomegalovirus strains from overlapping cosmid and plasmid clones

We have developed a cosmid and plasmid system to generate mutant strains of murine cytomegalovirus (MCMV). The system is based on a series of seven ov erlapping cosmid clones that regenerate MCMV when cotransfected into mouse cells, The unaltered cosmids produce MCMV that is indistinguishable from wi ld-type MCMV based on restriction enzyme digest patterns of virus DNA and g rowth rates both in vitro and in vivo, Analysis of viral DNA from plaque-pu rified recombinant isolates taken from in vitro and in vivo stocks indicate d that regeneration did not introduce novel mutations in the recombinant vi ral genomes, Isolation of specific genes and subsequent generation of speci fic mutant MCMVs was accomplished by replacement of cosmids with overlappin g plasmid subclones, A new vector, PmeSUB, featuring a multiple cloning sit e and a stringent origin of replication, was constructed to make large subc lones for use with smaller subclones containing the gene of interest. The u tility of this system was demonstrated by the generation of two different m utant MCMVs from different combinations of overlapping plasmid subclones of one cosmid, The advantages of this system are that (i) target genes are ma intained as small clones making them amenable to standard in vitro mutagene sis manipulations and that (ii) no reporter or selection genes are necessar y to identify mutants.