Genetic analysis of cytomegalovirus by shuttle mutagenesis.
The genomes of Herpesviridae family members are among the largest of all viruses and therefore present a formidable challenge in understanding the roles of every gene in replication or pathogenesis. For example, murine cytomegalovirus (MCMV) has a genome of 230 kb that encodes more than 170 genes, many of which have unknown functions. Many techniques for the genetic analysis of a herpesvirus have been developed over the past two decades. One such procedure involves the use of a shuttle mutagenesis system, and it has successfully generated a pool of MCMV mutants that contained an engineered Tn3-type transposon inserted within their genome. The process of shuttle mutagenesis involves the construction of a genomic fragment library, transposon mutagenesis of the library, and generation of virus mutants through homologous recombination. This chapter details the methodologies required for implementing a Tn3-based shuttle mutagenesis system for construction of a mutant virus library.
Lee
Manfred
M
Division of Infectious Diseases, School of Public Health, University of California-Berkeley, Berkeley, CA, USA.
Liu
Fenyong
F
D003587:Cytomegalovirus / Q000235:genetics*; D004251:DNA Transposable Elements; D005822:Genetic Vectors; D015698:Genomic Library; D009154:Mutation; D010957:Plasmids
D004251:DNA Transposable Elements