Project description:Recombinant baculoviral vectors efficiently transduce several types of cells in the brain. To characterize host responses to viral challenge, thus verifying the suitability of using the virus for the development of gene therapy strategies in the central nervous system, we used cDNA microarray technology to examine in vitro and in vivo global cellular gene expression profiles after viral transduction. We demonstrated that the transduction induced host antiviral responses as a major reaction in all three types of samples profiled, including the rat brain, cultured human astrocytes and human neuronal cells. The related genes were mainly those associated with innate immunity. Several genes of the major histocompatibility complex molecules, an important component of the host adaptive immunity to exogenous pathogens, were up-regulated in the rat brain and human astrocytes, but not in neuronal cells. We also observed that genes related to cell death and apoptosis were up-regulated and genes related cell cycle regulation were down-regulated in neuronal cells, but not obviously affected in astrocytes. These findings should be useful in understating the molecular basis for neural cell response to baculoviral transduction and guiding rational applications of baculoviral vectors in the central nervous systems Experiment Overall Design: CMV-Luc baculovirus with a luciferase reporter gene under the control of hCMV promoter and CMV-GFP baculovirus with a gene encoding for a green fluorescence protein were constructed as described previously (Wang et al., 2006). Both vectors were used to confirm baculoviral transduction. Our pilot test, as well as previous studies (Stilwell and Samulski, 2003; Zhao et al., 2005), demonstrated no detectable difference between viral vectors with and without a reporter gene in affecting global gene expression profiles. Thus, CMV-Luc baculovirus was used throughout microarray experiments. Experiment Overall Design: Recombinant baculovirus vectors were produced and propagated in Spodoptera frugiperda (Sf9) insect cells according to the manual of the Bac-to-Bac baculovirus expression system (Invitrogen). Sf9 insect cells pre-adapted to Sf-900 II serum-free medium were purchased from Invitrogen (Carlsband, CA) and grown in spinner flasks at 27.5oC. Budded viruses in the insect cell culture medium were collected and filtered through a 0.45-Î¼m pore size filter (Minipore, Bedford, MA, USA) to remove any contamination, and concentrated by ultracentrifugation at 28,000g for 60 min. Viral pellets were re-suspended in appropriate volumes of 0.1 M phosphate-buffered saline (PBS) and their infectious titers (plaque-forming units, pfu) were determined by plaque assay on Sf9 cells. Experiment Overall Design: For in vitro study, normal human astrocytes (Lonza, Basel, Switzerland) were cultured in Astrocyte Basal Medium (ABM) supplemented with the AGM SingleQuots. Cells were maintained according to manufacturerâs instructions. Human neuronal cells, transdifferentiated from human Cord Blood Stem Cell culture, were obtained from Celprogen (San Pedro, CA). The cells were cultured on flasks coated with neuronal expansion matrix in human neuronal expansion complete media and maintained according to the manufacturerâs instructions. Cells were transduced with baculoviruses in Optimem (Invitrogen) at an MOI of 50 and incubated at 37Â°C for 1 h. After the incubation, the medium containing the viruses was replaced by fresh growth medium, and the cells were collected 48 hours later for analysis.

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Instability of WT AAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system

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