Generation of a transposon insertion mutant library for bovine herpesvirus 4 cloned as a bacterial artificial chromosome by in vitro MuA based DNA transposition system.
Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Although the BoHV-4 genome has been sequenced, the function of the majority of putative genes is elusive. Several features make BoHV-4 attractive as a backbone for use as a viral expression vector and/or as a model to study gamma herpesvirus biology and determining which genes are essential for its replication is a very important task. Starting from BoHV-4 genome cloned as infectious bacterial artificial chromosome (BAC-BoHV-4) in Escherichia coli. A random insertion mutant library for BoHV4 was generated by the use of MuA transposase-catalyzed in vitro transposition reaction. Viral mutant transfection and direct sequencing allow the rapid determination of which BoHV-4 genes are essential for viral growth in a permissive eukaryotic cell line. BoHV-4 functional analysis information is fundamental when the BoHV-4 genome is modified for vector purposes.
Donofrio
Gaetano
G
Università di Parma, Facoltà di Medicina Veterinaria, Dipartimento di Salute Animale, Sezione di Malattie Infettive degli Animali, via del Taglio 8, 43100 Parma, Italy. gaetano.donofrio@unipr.it
Martignani
Eugenio
E
Sartori
Chiara
C
Vanderplasschen
Alain
A
Cavirani
Sandro
S
Flammini
Cesidio Filippo
CF
Gillet
Laurent
L
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004279:DNA, Viral; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D026021:Herpesvirus 4, Bovine / Q000235:genetics*; D066298:In Vitro Techniques; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D019895:Transposases / Q000378:metabolism
D004251:DNA Transposable Elements; D004279:DNA, Viral; D019895:Transposases