Cloning of complete genomes of large dsDNA viruses by in vitro transposition of an F factor containing transposon.
An improved bacmid technology for cloning complete genomes of large dsDNA viruses with circular genomes has been developed and tested. The system, termed EZ::BAC, is based on Escherichia coli F factor replicon, a chloramphenicol resistant marker gene with the mosaic ends recognized specifically by the transposase of the Tn5. In vitro transposition was carried out for the baculovirus shuttle vector pMON14272 (136kb) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome (134kb) as target DNAs. Transposon EZ::BAC was inserted randomly into the target DNAs, leading to 9bp duplication of the flanking end at the insertion site. One of the obtained AcMNPV::BACs replicated in Sf21 cells after transfection. The random in vitro generation of viral bacmids using EZ::BAC facilitates the host-independent propagation of intact and functional viral genomes in E. coli cells and does not require sequence information of the target DNA as is necessary for the generation of bacmids in conventional systems.
Wang
Yongjie
Y
Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Centre Palatinate (DLR Rheinpfalz), Breitenweg 71, 67435 Neustadt an der Weinstrasse, Germany.
Stojiljković
Nina
N
Jehle
Johannes A
JA
D000818:Animals; D002460:Cell Line; D002702:Chloramphenicol Resistance; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004251:DNA Transposable Elements; D004267:DNA Viruses / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005144:F Factor; D005822:Genetic Vectors; D016679:Genome, Viral; D018411:Spodoptera
D004251:DNA Transposable Elements; D004270:DNA, Circular; D004247:DNA