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Abstract Background Baculovirus expression vector system (BEVS) has become a standard in recombinant protein production and virus-like particle preparation for numerous applications. Findings We describe here protocols which adapt baculovirus generation into 96-well format. Conclusion The established methodology allows simple baculovirus generation, fast virus titering within 18 h and efficient recombinant protein production in a high-throughput format. Furthermore, the produced baculovirus vectors are compatible with gene expression in vertebrate cells in vitro and in vivo.

Background Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. Methods Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-DΔNΔC was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. Results A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding β-galactosidase ( lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 109 plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. Conclusions We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-DΔNΔC as a therapeutic transgene. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

An abstract of the article "A Multipurpose Vector System for the Simultaneous Screening of Libraries in Bacteria, Insect and Mammalian Cells and Expression In Vivo," by Olli H. Laitinen, Kari J. Airenne, and Erik Peltomaa is presented.

Human mesenchymal stem cells (hMSCs) are used in applications, which may require a large amount of cells; therefore, efficient expansion of the cells is desired. We studied whether TiO2 coating on plastic cell culture dishes could promote proliferation of hMSCs without adverse effects in chondrogenic differentiation. TiO2-films were deposited on polystyrene dishes and glass coverslips using an ultrashort pulsed laser deposition technique. Human MSCs from three donors were expanded on them until 95% confluence, and the cells were evaluated by morphology, immunocytochemistry and quantitative RT-PCR (qRT-PCR). The chondrogenic differentiation in pellets was performed after cultivation on TiO2-coated dishes. Chondrogenesis was evaluated by histological staining of proteoglycans and type II collagen, and qRT-PCR. Human MSC-associated markers STRO-1, CD44, CD90 and CD146 did not change after expansion on TiO2-coated coverslips. However, the cell number after a 48h-culture period was significantly higher on TiO2-coated culture dishes. Importantly, TiO2 coating caused no significant differences in the proteoglycan and type II collagen staining of the pellets, or the expression of chondrocyte-specific genes in the chondrogenesis assay. Thus, the proliferation of hMSCs could be significantly increased when cultured on TiO2-coated dishes without weakening their chondrogenic differentiation capacity. The transparency of TiO2-films allows easy monitoring of the cell growth and morphology under a phase-contrast microscope.

Baculovirus expression vector system (BEVS) is well known as a feasible and safe technology to produce recombinant (re-)proteins in a eukaryotic milieu of insect cells. However, its proven power in gene delivery and gene therapy is still poorly recognized. The basis of BEVS lies in large enveloped DNA viruses derived from insects, the prototype virus being Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Infection of insect cell culture with a virus encoding a desired transgene under powerful baculovirus promoter leads to re-protein production in high quantities. Although the replication of AcMNPV is highly insect specific in nature, it can penetrate and transduce a wide range of cells of other origin. Efficient transduction requires only virus arming with an expression cassette active in the cells under investigation. The inherent safety, ease and speed of virus generation in high quantities, low cytotoxicity and extreme transgene capacity and tropism provides many advantages for gene delivery over the other viral vectors typically derived from human pathogens.

Baculoviruses are safe and high-capacity vectors for gene delivery which have matured from the initial successful experiments performed in liver cells into convenient tools to transduce almost any cell from any origin in vitro and ex vivo. This is a result of 15 years of intensive vector development as well as studies performed in vertebrate cells to reveal important factors affecting the transduction efficacy. Now, at the stage when the first evidence of meaningful use of baculoviruses for therapeutic applications has been reported, there is no doubt that the technology will meet the expectations as highly useful platform for many applications of gene delivery. This review summarizes the pre-clinical in vivo work carried out with baculoviruses and discusses remaining challenges which still need to be solved.