Early detection of productive baculovirus DNA transfection.

The most widely studied baculovirus, the Autographa californica M nucleopolyhedrovirus (AcMNPV), is a large, enveloped DNA virus that infects lepidopteran larvae (8). This virus is commonly used as a eukaryotic vector to express heterologous genes in cultured insect cells and insect larvae (1,3,15). Baculovirus expression systems offer several advantages over other expression systems, among which are the capacity for large DNA insertions, high levels of recombinant protein synthesis, production of proteins that are similar in biological activity, stability, posttranslational modifications (e.g., glycosylation, phosphorylation, etc.), structure and immunological activity, as compared to naturally occurring proteins (2,7,14,15). In some cases, the recombinant proteins are targeted to appropriate cellular compartments (1). Since the protective protein occlusion matrix (polyhedrin) is nonessential for AcMNPV propagation in cell culture, the polyhedrin gene in the baculovirus expression vector systems is usually replaced by heterologous genes that are overexpressed under the transcriptional control of the strong polyhedrin promoter, which is activated at later stages of the viral replication cycle (10). The early systems relied on substitution of the polyhedrin gene with the foreign gene by homologous recombination, between wild-type virus DNA and the heterologous gene construct after co-transfection of these DNAs into insect cells. In this case, the number of recombinant plaques is usually low, requiring sequential plaque assays and viral amplification to purify recombinant virus and obtain sufficient amount of virus for protein expression (1,7,9,15). Also, it is difficult even for experienced researchers to distinguish occlusion-minus recombinant plaques from noninfected cells and to monitor cell-density decreases and cell-size increases in cultures infected with recombinant baculovirus (2). The recently developed BAC-TOBAC system (Life Technologies, Rockville, MD, USA) is based on sitespecific transposition of an expression cassette from a plasmid (pFAST-BAC) to a baculovirus shuttle vector (bacmid) that is propagated in E. coli. The foreign gene is expressed after transfection of the recombinant bacmid DNA into insect cells, resulting in a recombinant baculovirus that may be used to infect other insect cell cultures (1,5,9,11, 14). This improvement allows a more rapid production of the recombinant protein without isolation of viral plaques. However, since the polyhedrin gene has been deleted from the bacmid, only subtle morphological differences can be detected between infected and uninfected cells using either parental or recombinant baculovirus, making it difficult to confirm productive transfection or infection. To distinguish between infected and uninfected cells, some markers have been proposed such as β-galactosidase, luciferase and fluorescent proteins (2,15). These markers are effective, but they rely on specially constructed vectors and specific assays that can be cumbersome and time consuming. They also generate fusion proteins, which may not be desirable. Based on our experience with animal viruses, we propose two rapid and practical methods that allow confirmation of productive transfection with bacmid DNA: (i) subculture of the transfected Spodoptera frugiperda (Sf9) cells that leads to a dramatic amplification of the cytopathic effect; and (ii) PCR amplification of the recombinant baculovirus DNA present in culture supernatants from the first virus amplification. These methods allow early detection of any problem in the bacmid transfection, thus avoiding the waste of time and reagents involved in subsequent steps. Results presented here were obtained using recombinant baculovirus constructs encoding the complete sequence of the glycoprotein C (gC) gene from bovine herpesvirus 1 (BHV-1). Two different inserts were separately subcloned into the pFASTBAC HT vector (Life Technologies): a 2.4-kb NcoI/EcoRI fragment containing the complete sequence of gC, and a 1.3-kb NcoI/SalI fragment containing a partial sequence of gC that lacks the transmembrane terminal portion (manuscript in preparation). The resulting plasmids (pFASTBAC-HT-gC and pFASTBAC-HT-partial gC) were prepared by alkaline lysis DNA purification (12). Recombinant plasmids were transformed into DH10BAC-competent bacteria (Life Technologies), and white colonies (with transposed recombinant bacmid) were selected and grown overnight for recombinant bacmid DNA purification, according to the BAC-TO-BAC baculovirus expression system instructions. Correct transposition of gC sequences from pFASTBAC HT into the bacmid was confirmed by automated DNA sequencing. Bacmid DNA isolation and cell transfection were performed as described in the BAC-TO-BAC manual. Recombinant bacmid DNA was isolated from 1.5-mL cultures using the alkaline lysis protocol. The presence and integrity of bacmid DNA in these preparations were analyzed by electrophoresis of 4 μL of each miniprep through a 0.8% agarose gel in 1× TAE (12) and stained with ethidium bromide. Bacmid DNA was directly used to transfect Sf9 cells. Transfection of 9 × 105 Sf9 cells/35mm dish was performed using 5 μL recombinant bacmid DNA and 6 μL CellFECTIN reagent in 1 mL SF900 II SFM reagent (both from Life Technologies) without antibiotics. After 5 h, the medium was changed to 2 mL TNMFH medium (Grace’s insect cell medium supplemented with 4 g/L yeastolate, 3.3 g/L lactoalbumin hydrolysate (Life Technologies) plus 10% fetal calf serum (Cultilab, Campinas, Brazil), and cells were maintained at 28°C. Untransfected cells and cells transfected with empty bacmid DNA (without transposed DNA) were used as transfection controls. At 72 h after transfection, the medium was recovered, clarified by centrifugation at 800× g for 5 min and stored as viral stocks, protected from light at 4°C. After collection of transfected Sf9 cell medium, we suggest subculturing the transfected cells (a blind passage) as a method to rapidly amplify viral titers and enhance the cytopathic effect caused by the recombinant baculovirus generated during transfection. The cells from each transfected culture were resuspendBenchmarks