Gene Expression Changes in Response to Baculoviral Vector Transduction of Normal Human Astrocytes In Vitro
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE9834 | GEO | 2007/12/13
SECONDARY ACCESSION(S): PRJNA103807
REPOSITORIES: GEO
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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 Keywords: Baculovirus, brain, astrocytes, neurons, transduction, Infection, gene expression analysis, DNA microarray
2007-12-13 | GSE9835 | GEO
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 Keywords: Baculovirus, brain, astrocytes, neurons, transduction, Infection, gene expression analysis, DNA microarray
2007-12-13 | GSE9833 | GEO
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 Keywords: Baculovirus, brain, astrocytes, neurons, transduction, Infection, gene expression analysis, DNA microarray
2009-04-17 | GSE14484 | GEO
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-μm 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 37°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.
2008-06-17 | E-GEOD-9834 | biostudies-arrayexpress
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-μm 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 37°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.
2008-06-17 | E-GEOD-9835 | biostudies-arrayexpress
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: For in vivo viral inoculation, adult male Wistar rats (weighing 250â300 g) were used. Five μl of baculovirus vectors (5 x 107 viral particles) was injected stereotaxically into the striatum at two injection sites (AP ±1.0mm, ML +2.5mm, and DV -5.0mm from bregma and dura) using a 10 μl Hamilton syringe connected with a 30-gauge needle, at a speed of 0.5 μl/min. The needle remained in place for another 5 min before being slowly retracted. Rats were sacrificed and the brain tissues were collected 48 hours later for analysis. Five μl of PBS was injection into the contralateral striatum and the tissues were collected and used as controls. Experiment Overall Design: Seven rats were used for the study. Experiment Overall Design: Total RNA was isolated using Trizol® (Invitrogen, Carlsbad,CA) according to the manufacturerâs instruction. RNA was further cleaned up using RNeasy mini kit (Qiagen). Total RNA was quantified using a Nanodrop spectrophotometer and its quality was further assessed using an RNA nanochip on a Bioanalyzer. One to 10 ïg of good quality RNA (i.e. A260/A280 > 2.0 and A230/A260 > 2.1) was used to prepare labeled cRNA using either Affymetrixâs One-Cycle Target Labeling and Control Reagents or Kreatechâs RNA ampULSe amplification and labeling kit (Kreatech, Amsterdam, The Netherlands). Hybridization mixes with 15 ïg of fragmented cRNA previously checked on the bioanalyzer were prepared according to Affymetrixâs GeneChip® Hybridization, Wash, and Stain Kitâs instruction and used to hybridize the human GeneChipï¢ Human Genome U133 Plus 2.0 or GeneChipï¢ Rat Genome 230 2.0 for 16 hours at 45ºC. The arrays were washed and stained with phycoerythrin according to the manufacturerâs instructions. Chips were scanned using GeneChip® Scanner 3000 and data acquired using the GCOS software. Experiment Overall Design: Data obtained from GCOS was exported using the Data Transfer Tool and transferred to GeneSpring and Genedata software respectively. The GC-RMA preprocessor file was used to re-process imported CEL files and to normalize the data at the probe level. Replicates (at least 3 per experiment) were grouped and data normalized using the default setting of Genespring (Data transformation: values less than 0.01 to 0.001; Global normalization function: Per Chip normalization to median or a percentile; Gene normalization function: normalize to median was used. The Genedataâs Refiner function was used to assess the overall quality of the hybridization. To analyze the data, the genes were grouped according to the cellsâ or brainâs treatment (Non- infected/infected) and the log of normalized values were taken for further analysis. Data was filtered according to their flags in such a way that at least 5 out of 7 brain samples or 2 out of 3 cell replicates had a present call and that at least one of the groups of replicates (non-infected/infected) fulfilled this condition. The volcano plot was sometime used to filter the data further with the following cut-off (2-fold difference in expression level and t-test p-value of less than 0.05). Further data manipulation for comparison purposes are described in the text.
2008-06-16 | E-GEOD-9833 | biostudies-arrayexpress
Project description:Recombinant insect baculoviral vectors efficiently transduce several types of cells in the brain and can possibly be used for gene therapy for brain disorders. To verify the suitability of using these viral vectors to develop gene therapy strategies in the brain, we evaluated immune reactions upon acute administration of baculoviral vectors into the brain of the cynomolgus macaque using microarray global gene expression profiling.
2015-11-21 | GSE75264 | GEO
Project description:Recombinant insect baculoviral vectors (BV) efficiently transduce several types of cells in the brain and can possibly be used for gene therapy for brain disorders. To verify the suitability of using these viral vectors to develop gene therapy strategies in the brain, and to evaluate our method of virus purification, we evaluated immune reactions upon acute administration of BV that were purified by ion-exchange membrane chromatography with high-speed centrifugation or high-speed centrifugation alone into the mouse brain using microarray global gene expression profiling. Adult male mice (Mus musculus) were administered with baculoviral vectors purified by membrane chromatography with high-speed centrifugation (MC+HS) or baculoviral vectors purified by high-speed centrifugation (HS) alone into the brain. We sought to compare the gene expression changes in the brain triggered by MC+HS-purified and HS-purified baculoviral vectors.
2015-11-21 | E-GEOD-75265 | biostudies-arrayexpress
Project description:Recombinant insect baculoviral vectors efficiently transduce several types of cells in the brain and can possibly be used for gene therapy for brain disorders. To verify the suitability of using these viral vectors to develop gene therapy strategies in the brain, we evaluated immune reactions upon acute administration of baculoviral vectors into the brain of the cynomolgus macaque using microarray global gene expression profiling. Adult male cynomolgus macaques (Macaca fascicularis) were administered with baculovirus BV-HSVtk purified by membrane chromatography + high-speed centrifugation (MC+HS) into the brain.
2015-11-21 | E-GEOD-75264 | biostudies-arrayexpress
Project description:Recombinant insect baculoviral vectors (BV) efficiently transduce several types of cells in the brain and can possibly be used for gene therapy for brain disorders. To verify the suitability of using these viral vectors to develop gene therapy strategies in the brain, and to evaluate our method of virus purification, we evaluated immune reactions upon acute administration of BV that were purified by ion-exchange membrane chromatography with high-speed centrifugation or high-speed centrifugation alone into the mouse brain using microarray global gene expression profiling.
2015-11-21 | GSE75265 | GEO