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ABSTRACT:
PROVIDER: PRJNA884178 | ENA |
REPOSITORIES: ENA
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Project description:The human Adeno-Associated Virus serotype 2 (WT AAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. However, it has been shown that WT AAV2 and recombinant AAV display significantly different characteristics, especially regarding infection rate, with a near perfect infectivity and better encapsidation rate of WT AAV2. Even though rAAVs are routinely produced in the Baculovirus/Sf9 cell system, WT AAV2 has never been produced in this context. To understand the infectivity and encapsidation rate differences between WT AAV2 and rAAV, we tried to produce WT AAV2 in baculovirus/Sf9 cells system hypothesizing that the WT AAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce WT AAV2 in Baculovirus/Sf9, we found that WT AAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system. p5 promoter activity in the baculovirus/Sf9 cell system led to the expression of Rep78 that finally excises WT AAV2 genome from the baculovirus genome during the earliest phase of baculovirus stock production. The p5 promoter expression kinetics and the specific strand RNA-Seq analysis of the WT AAV2, rAAV Rep2/Cap2 cassettes in the baculovirus context was performed. We demonstrate that the WT AAV2 native promoters, p5, p19 and p40 are all active and lead to the expression of different proteins and peptides. In addition, this study demonstrates that the baculovirus brings at least some of the helper functions needed in the AAV replication/life cycle.
2018-06-21 | GSE96855 | GEO
Project description:Baculovirus expression systems have been widely used to produce recombinant mammalian proteins owing to the lack of viral replication in vertebrates. Although several lines of evidence have demonstrated impacts of baculovirus infection in mammalian hosts, genome-wide effects have not been fully elucidated. Here, we provide comparative transcriptome profiles of baculovirus and host-immune response genes in recombinant baculovirus-infected mammalian and insect cells. Specifically, to decipher the impacts of baculovirus infection in mammalian cells, we conducted total RNA-seq on human 293 cells and insect Sf9 cells infected with recombinant baculovirus. We found that baculovirus genes were rarely expressed under the control of baculoviral promoters in 293 cells. Although some baculovirus early genes, such as PE38 and IE-01, showed limited expression in 293 cells, baculoviral late genes were mostly silent. We also found modest induction of a small number of mammalian immune response genes associated with Toll-like receptors, cytokine signaling, and complement in baculovirus-infected 293 cells. These comprehensive transcriptome data will contribute to improving recombinant baculovirus as tools for gene delivery, gene therapy, and vaccine development.
2020-09-28 | GSE158603 | GEO
Project description:KKT23 is a kinetoplastid kinetochore protein that has structural similarity to the GCN5 acetyltransferase domain. We performed crosslinking mass spectrometry of the recombinant KKT3-KKT22-KKT23 complex purified from Sf9 insect cells.
2024-08-19 | PXD052923 | Pride
Project description:Settlement-inducing protein complex (SIPC) is a protein that acts as a phoromone cue to attract conspecific of the barnacle Amphibalanus amphitrite to settle on a substrate. Recombinant settlement-inducing protein complex was expressed in insect Sf9 cells through a baculovirus overexpression system, purified and analysed by mass spectrometry to confirm its identity.
2019-01-07 | PXD006858 | Pride
Project description:Small nucleolar RNAs (snoRNA) function in guiding 2'-O-methylation and pseudouridylation of ribosomal RNAs. But we found that knock down of a C/D box snoRNA, Bm-15, can induce apoptosis of insect Spodoptera frugiperda Sf9 cells. For the genome sequence of Spodoptera frugiperda is incomplete, here with the de novo sequencing method, transcriptome of Spodoptera frugiperda cell line Sf9 were sequenced after being transfected with overexpression vector and repression probes of snoRNA Bm-15. Results showed that 21 apoptosis-related genes were up-regulated upon Bm-15 inhibition and down-regulated with Bm-15 overexpression.
2019-04-17 | GSE98196 | GEO
Project description:Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV-manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5 producing HEK293 cells using reversed phase nano liquid chromatography and tandem mass spectrometry (LC-MS/MS). Rela-tive label-free quantitation (LFQ) was performed allowing a comparison of transfected vs. un-transfected cells. Gene ontology enrichment and pathway analysis revealed differential expres-sion of proteins involved in fundamental cellular processes such as metabolism, proliferation and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells thus potentially enabling further improvements of gene therapy product manufacturing.
2021-12-09 | PXD028154 | Pride
Project description:The phosphorylation profile of Sgk3 protein expressed either in Sf9 insect cells or HEK 293T human cells was assessed by mass spectrometry.
2022-02-17 | PXD026281 | Pride
Project description:Characterization of Residual MicroRNAs in AAV Vector Batches Produced in HEK293 Mammalian Cells and Sf9 Insect Cells
| PRJNA1127999 | ENA
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