Use of bacterial artificial chromosomes in generating targeted mutations in human and mouse cytomegaloviruses.
Cloning of cytomegalovirus (CMV) genomes as bacterial artificial chromosomes (BAC) in E. coli and their manipulation using the techniques of bacterial genetics has greatly facilitated the construction of CMV mutants. This unit describes easily applicable procedures that allow rapid introduction of any kind of targeted mutation into BAC-cloned CMV genomes. Protocols for the reconstitution of virus from isolated BAC DNA, preparation of a virus stock, and isolation and characterization of viral DNA are also included. Special emphasis is laid on description of critical steps and thorough characterization of the altered BACs.
Borst
Eva Maria
EM
Institute of Virology, Hannover Medical School, Hannover, Germany.
Benkartek
Corinna
C
Institute of Virology, Hannover Medical School, Hannover, Germany.
Messerle
Martin
M
Institute of Virology, Hannover Medical School, Hannover, Germany.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification* / Q000472:pathogenicity; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005821:Genetic Techniques; D016679:Genome, Viral; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D014169:Transformation, Bacterial / Q000235:genetics; D014774:Virulence / Q000235:genetics
D004279:DNA, Viral; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase