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Pseudorabies virus (PRV), an important pathogen causing Aujeszky's disease in swine and leading to latent infection (6), is a member of the Alphaherpesvirinae subfamily and could served as a useful model organism for the study of herpesvirus biology (9). Like most of the herpesviruses, PRV has a very large double stranded DNA genome (~143kb) with more than 70 genes identified (3). Functional characterization of these viral genes, by generating null mutants and investigating the resulting changes in phenotype, is very important in understanding the molecular aspects of herpesvirus replication and pathogenesis. For some key genes, smaller targeted mutations are required to identify the distinct functional domains within the proteins (9).
Such studies often require establishment of large numbers of recombinant viruses which are usually created by homologous recombination in infected cells relying on the cellular recombination and repair machinery. However, this can be a laborious and sometimes impossible task, especially if the mutant has a severe growth disadvantage compared to the wild-type virus. Bacterial artificial chromosomes (BACs), single copy F-factor-based plasmid vectors of intermediate insert capacity (15), have now enabled the cloning of complete herpesvirus genomes and infectious virus genomes can be shuttled between Escherichia coli (E. coli) and eukaryotic cells. While herpesvirus BAC DNA engineering in E. coli requires neither restriction sites nor cloning steps and allows the introduction of a wide variety of DNA modifcations, the large size of these bacmids precludes the use of rapid in vitro methods of manipulation commonly employed for construction of small plasmids.
Tn7, a site-specific transposon, transposes almost exclusively to a distinct attachment site named attTn7 within the E. coli genome (1). By introducing this attTn7 sequence into a BAC, Tn7 can serve as an insertion vehicle (4). This is particularly useful if numerous genes and constructs need to be tested for their expression in the context of viral genome. Tn7-mediated transposition has been well exploited for research on functional genomics of baculoviruses (4). Recently, this technology has been applied to bacmid-cloned cytomegalovirus (CMV), a member of the Gammaherpesvirinae subfamily, for rapid recombinant virus construction (2).
In this paper, we report the development of a technology employing Tn7-mediated transposition as a rapid and reliable method for recombinant PRV construction. A lacZα-mini-attTn7 region was inserted into the intergenic region between the gG and gD genes in an attempt to maintain every gene and element of the parental virus. Then green fluorescent protein (GFP) gene was introduced to test the utility of this transposition system and the stability of mini-Tn7 insertions in cell culture. The technology should greatly facilitate the detailed mutagenic studies of PRV.
Tn7-mediated Introduction of DNA into Bacmid-cloned Pseudorabies Virus Genome for Rapid Construction of Recombinant Viruses*
- Received Date: 04 April 2007
- Accepted Date: 18 May 2007
Abstract: lacZα-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome (BAC) by homologous recombination in E. coli. The resulting recombinant BAC (pBeckerZF1) was confirmed by PCR and sequencing. Green fluorescent protein (GFP) gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect (CPE) observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3. vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.