Rapid identification of non-essential genes for in vitro replication of Marek's disease virus by random transposon mutagenesis.
Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. The MDV genome encodes more than 100 genes. However, the role of many of these genes in virus replication is not known. The construction of an infectious bacterial artificial chromosome (BAC) clone of the highly oncogenic RB-1B strain of MDV has been described previously. Virus reconstituted from the BAC clone induced rapid-onset lymphomas in chickens very similar to the wildtype viruses. In this paper, the construction of a high-density random transposon-insertion mutant library of the RB-1B BAC clone using a high throughput in vitro transposon mutagenesis technique is described. Furthermore a PCR screening method, using primers specific for the transposon sequence and the MDV gene(s) of interest, was developed for the rapid identification of specific insertion mutants. The application of the screening method to identify some of the non-essential genes for MDV replication in vitro is described.
Chattoo
Jason P
JP
Division of Microbiology, Institute for Animal Health, Compton, Berkshire RG20 7NN, United Kingdom.
Stevens
Mark P
MP
Nair
Venugopal
V
D022202:Chromosomes, Artificial, Bacterial; D004251:DNA Transposable Elements; D025922:Mardivirus / Q000235:genetics* / Q000502:physiology; D016296:Mutagenesis; D016366:Open Reading Frames; D014779:Virus Replication
D004251:DNA Transposable Elements