Baculoviruses and mammalian cell-based assays for drug screening.
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Animals↗
Biomedical subjects
Thomas A Kost
Publications and source records attributed to Thomas A Kost.
Baculovirus as versatile vectors for protein expression in insect and mammalian cells.
Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.
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A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry.
A cell-based time-resolved fluorescence (celTRF) immunoassay is described for pre-screening antibodies to G protein-coupled receptor (GPCR) peptides that predicts suitability for immunohistochemistry (IHC). Rat GPCRs were expressed in Saos-2 human osteosarcoma cells via recombinant baculoviruses designed for mammalian cell expression, i.e., the transduced cells were used as a "screening lawn". The lawn was fixed and permeabilized similarly to IHC tissue. The celTRF, a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), employed Eu-labelled goat anti-rabbit IgG. It exhibited a broad dynamic range upon which enzyme-linked immunosorbant assay (ELISA)-positive affinity-purified anti-peptide antibody reagents were examined for specificity and potency. Over 150 anti-peptide reagents to 27 GPCRs were characterized. All celTRF-positive antibodies were found to be suitable for IHC, whereas ELISA alone did not predict IHC utility. Examples are illustrated with five rabbit anti-neuropeptide FF receptor 1 (NPFF1) antibodies, where a strong correlation between celTRF potency and IHC utility was observed in both applications. In contrast, two high anti-peptide ELISA titer but celTRF-negative antibodies failed to recognize the NPFF1 receptor in IHC. The celTRF assay was performed manually and in an automated fashion, in our case, using a Biomek FX station and Sami scheduling software. The celTRF is the first in vitro automated assay that offers confident pre-selection of antibodies for IHC and the versatility to accommodate the rapid screening of large numbers of GPCRs. The celTRF is readily applicable to other protein target classes.
Antibodies↗
Influence of promoter choice and trichostatin A treatment on expression of baculovirus delivered genes in mammalian cells.
The expression of recombinant proteins following transduction of CHO cells with recombinant baculoviruses containing a mammalian expression cassette with the CMV-promoter is enhanced by the addition of trichostatin A (TSA), a specific histone deacetylase inhibitor. To further investigate the effect of TSA treatment on protein production following BacMam transduction, viruses containing various viral promoters (SV40, CMV, and RSV) and one cellular promoter (human ubiquitin C) were compared with regard to expression level of a gfp-luciferase fusion protein following transduction of CHO, COS-1, and HEK293 cells. The overall effect on expression appears to be cell specific, indicating that different mechanisms are active within different cell lines. Further, COS cells transfected with naked viral DNA, plasmids, and baculovirus particles were compared in regard to TSA treatment. The increase in reporter gene expression observed following BacMam transduction and TSA treatment were greater than those for transfection of either naked viral DNA or plasmid DNA.
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Identification of peptides that inhibit the DNA binding, trans-activator, and DNA replication functions of the human papillomavirus type 11 E2 protein.
Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPV-associated diseases.
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Baculovirus-mediated gene delivery into mammalian cells.
A relatively recent advance in the use of recombinant baculoviruses is their use for delivery of genes and genetic elements into mammalian cells. Baculovirus vectors retrofitted with mammalian gene promoters have been shown to efficiently deliver and express genes in a broad assortment of cell types. These baculovirus transductions are simple to perform, reproducible, and demonstrate no overt cell toxicity. Baculovirus-mediated gene delivery is particularly useful for repetitive or moderately high-throughput procedures such as cell-based assays, or for situations where transfection procedures are inadequate.
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Recombinant baculoviruses used to study estrogen receptor function in human osteosarcoma cells.
We report that modified baculoviruses, termed BacMam viruses, can efficiently deliver multiple genes into mammalian cells to generate a heterologous transcription factor/reporter gene system. Using human estrogen receptor (ER) as a model nuclear receptor, we demonstrate how this approach can be successfully applied to assay development in Saos-2 human osteosarcoma cells. BacMam viruses containing full-length cDNAs were constructed for both human ER subtypes, ERalpha and ERbeta, and a third BacMam virus containing an ER-responsive reporter gene cassette. Using these viruses, we found that BacMam-ER expression/reporter constructs could be used to profile the effects of the agonist 17beta-estradiol and the partial agonist raloxifene in human Saos-2 cells. A comparison of assay data obtained with the BacMam-based system with that using standard DNA transfections demonstrates that the two systems are functionally equivalent, giving comparable EC(50) and IC(50) values for estrogen and estrogen plus raloxifene treatments, respectively. Our results indicate that BacMam-mediated gene transfer offers a novel and efficient method for delivery of nuclear receptors and associated genes for mammalian cell-based assay development.
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Recombinant baculoviruses as mammalian cell gene-delivery vectors.
The baculovirus expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, recombinant baculovirus vectors engineered to contain mammalian cell-active promoter elements, have been used successfully for transient and stable gene delivery in a broad spectrum of primary and established mammalian cells. The application of modified baculoviruses for in vivo gene delivery has also been demonstrated. In contrast to other commonly used viral vectors, baculoviruses have the unique property of replicating in insect cells while being incapable of initiating a replication cycle and producing infectious virus in mammalian cells. The viruses can be readily manipulated, accommodate large insertions of foreign DNA, initiate little to no microscopically observable cytopathic effect in mammalian cells and have a good biosafety profile. These attributes will undoubtedly lead to the increased application and continued development of this system for efficient gene delivery into mammalian cells. Who said you can't teach an old dog new tricks?
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Biotechnology. Paper Alert.
A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in biotechnology.
Biotechnology↗
Viruses as gene delivery vectors: application to gene function, target validation, and assay development.
A Biochemical Pharmacology Discussion Group Conference, was held at the headquarters of the New York Academy of Sciences on December 4, 2001 as part of an ongoing series designed to highlight and review areas important to modern drug development (Figure 1). Briefly introduced by Tom Kost (GlaxoSmithKline) and Michael Lotze (University of Pittsburgh), the focus was on the intersection of genomics, proteomics, and now "viromics." The latter term refers to the use of viruses and viral gene transfer to explore the complexity arising from the vast array of new targets available from the human and murine genomes. Indeed, access to large numbers of genes using viral vectors is a key tool for drug discovery and drug delivery. With 38,000 genes identified within the human genome, only 5000 are considered readily druggable. Generating tools such as these to validate targets represents a major part of the armamentarium of the postgenomic scientist. During the last 12 years alone, there have been over 26,000 publications on virus vectors. Many of them have been found useful in target validation, assay development, and evaluation in in vivo models and gene therapy. Thus, there is now an extensive knowledge base for several viral vectors, with unique attributes within each of them providing versatility, efficiency, and ease of use. The individual scientists presenting at the meeting illustrated many of the unique and useful characteristics of such vector systems including retrovirus, adenovirus, herpes virus, simbis virus, and baculovirus.
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