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sindbis virus vector

Tmem119 levels were unaffected at 24 h, however, showed a significant increase at 72 h. Astrocyte markers such as Gfap, S100 beta, vimentin and Aldh1a1 that are associated with reactive astrocytes were decreased. However, these polyproteins remained detectable in organotypic slices up to 72 h post-injection (Table 4), indicating viral vector particles were still present in organotypic slices. USA.gov. (2016). Trends Biotechnol. In Finland, antibodies against Sindbis virus were also detected in a high percentage amongst game birds, ... Recombination of replicon and helper RNAs and the emergence of propagation-competent vectors upon Sindbis virus vector production, International Journal of Molecular Medicine, 10.3892/ijmm.2013.1395, 32, 2, (410-422), (2013). Neurosci. the column was back equilibrated to the initial condition of 5% acetonitrile for 10 min. SIN virus is maintained predominately in wild bird … Next, peptides were fractionated on a Alltima C18 homemade column (300 μm id, 3 μm particle size), using a linear gradient of increasing concentration of mobile phase B (99.9% acetonitrile and 0.1% fluoroacetic acid) from 5% to 22% in 88 min, to 25% at 98 min, 40% at 108 min, and to 95% at 110 min.  |  Interferonv induced ifit proteins: their role in viral pathogenesis. 74. The microarray datasets are available upon request. Sample complexity was reduced by separating 30 μg of protein per sample on molecular weight using a 10% SDS polyacrylamide gel. To examine whether Sindbis viral vectors influence gene expression in hippocampal neurons, we prepared cultured organotypic slices of hippocampi from C57BL/6 mice. It is known to cause disease in animals including humans. 2014 Jun 15;23(12):1301-15. doi: 10.1089/scd.2013.0621. doi: 10.1074/mcp.O115.050229, Reinders, N. R., Pao, Y., Renner, M. C., da Silva-Matos, C. M., Lodder, T. R., Malinow, R., et al. Sindbis transduction did not affect the intrinsic electrophysiological properties of individual neurons: the membrane potential and neuronal excitability were similar between transduced and non-transduced CA1 neurons up to 72 h after Sindbis injection. |, Creative Commons Attribution License (CC BY). Mol. This may indicate that the exposure to Sindbis vectors triggers apoptotic cell death in cultured slices. A simple method for organotypic cultures of nervous tissue. (2016). Organotypic hippocampal slices were perfused with ACSF (in mM: 118 NaCl, 2.5 KCl, 26 NaHCO3, 1 NaH2PO4, 4 MgCl2, 4 CaCl2, and 20 glucose) gassed with 95% O2/5% CO2. Organotypic slices of the mouse hippocampus were injected with Sindbis viral vector expressing eGFP (50–100% of cells GFP+) or control-treated, and at 24 or 72 h post-injection total protein fractions were isolated, in-gel trypsin digested and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (Figure 2A). Specifically, each hybridization consisted of two individual samples from the same mouse, one transduced with Sindbis and one sham-treated and collected at the same time point post-injection. doi: 10.1016/S0896-6273(03)00124-7, Kebschull, J. M., Garcia da Silva, P., Reid, A. P., Peikon, I. D., Albeanu, D. F., and Zador, A. M. (2016). Transduction of neurons with a recombinant, replication-deficient Sindbis viral vector is a method of choice for studying the effects of short-term protein overexpression on neuronal function. US5217879A US07/815,602 US81560291A US5217879A US 5217879 A US5217879 A US 5217879A US 81560291 A US81560291 A US 81560291A US 5217879 A US5217879 A US 5217879A Authority US United States Prior art keywords sindbis virus sequence rna molecule nucleotides junction region Prior art date 1989 … It should facilitate the study of Sindbis and other RNA viruses. doi: 10.1038/nature04671, Stence, N., Waite, M., and Dailey, M. E. (2001). The gel was fixed overnight in fixation buffer (50% Ethanol and 28% Phosphoric Acid), washed three times in water and stained with Colloidal Coomassie Blue. BREDENBEEK,t ILYAFROLOV,CHARLESM. Methods 37, 173–182. Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. Gene Ontology analysis of genes that changed ≥2-fold significantly (FDR-corrected p < 0.05) upon 24 h of Sindbis-GFP treatment compared to control. 1997 Jun;7(3):289-98. doi: 10.1007/BF02740819. Current step recordings were analyzed with Clampex 10.7 (Molecular Devices). J. Neurosci. Error bars: SD. 19, 443–453. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the … Copy RNA was synthesized and hybridized to a mouse gene expression microarray containing 44.000 features (Figure 1A). Transduction of neurons with a recombinant, replication-deficient Sindbis viral vector is a method of choice for studying the effects of short-term protein overexpression on neuronal function. Post-mitotic neurons appear to be more tolerant toward recombinant Sindbis transduction: based on morphological and electrophysiological properties, hippocampal neurons transduced by Sindbis vectors remain viable for at least 48 h post-transduction (Ehrengruber et al., 1999; Maletic-Savatic et al., 1999; Marie et al., 2005; Kessels et al., 2009). Neuromodulation by a Cytokine: interferon-β differentially augments neocortical neuronal activity and excitability. The datasets generated for this study can be found in ProteomeXchange Consortium via the PRIDE partner repository, PXD013634. Sci. Recombinant Semliki forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. 39, 381–391. doi: 10.4103/1673-5374.226398, Kamenetz, F., Tomita, T., Hsieh, H., Seabrook, G., Borchelt, D., Iwatsubo, T., et al. After 8 min. Gene Ontology analysis of genes that changed ≥ 2-fold significantly (FDR-corrected p < 0.05) upon 72h of Sindbis-GFP treatment compared to control. The present invention relates to RNA vectors comprising Sindbis virus DI RNA having inserted therein heterologous RNA. (B) Comparisons in protein expression between Sindbis-GFP and control samples at 24h (left panel) and 72h (right panel). (1998). Neurochem. 733050106, dementia Research and Innovation Program “Memorabel” as a part of the “Deltaplan for Dementia.”. Brain Res. Infectious Sindbis virus vectors Download PDF Info Publication number US5217879A. Proteins that were only detected in Sindbis-GFP treated slices (top), or only detected in control treated slices (middle), or changed ≥2-fold significantly (FDR-corrected p < 0.05) upon Sindbis-GFP treatment compared to control (bottom). For instance, exposure of neurons to interferons can lead to an increased excitability or to enhanced synaptic currents (Calvet and Gresser, 1979; Vikman et al., 2001; Strauss et al., 2004; Stadler et al., 2014). All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Royal Netherlands Academy of Arts and Sciences (KNAW). Finally, because Sindbis virus is a blood-borne virus (31) that can cross the blood–brain barrier (15), Sindbis viral vectors should be able to reach most cells of the body. doi: 10.1039/b908315d, Deutsch, E. W., Csordas, A., Sun, Z., Jarnuczak, A., Perez-Riverol, Y., Ternent, T., et al. RNA integrity (average RNA integrity number 9.1, SEM 0.14, range 7.5–10) was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States). A., Piccini, A., Poncer, J. C., and Malinow, R. (2000). (C) Venn diagram showing the overlap between the transcripts that change levels more than 2-fold at 24 h and 72 h of Sindbis treatment compared to control treatment. Immuno-histochemical staining of tumor cells indicates that laminin receptor is elevated in tumor versus normal cells. Immunological aspects of microglia: relevance to Alzheimer’s disease. (2016). Transfection of this plasmid construct into mammalian cell lines (3T3, HepG2, and 293 cells) resulted in expression of the luciferase reporter gene. After hybridization, 43.020 features, encompassing 33.274 unique identifiers passed our detection criteria to be included in the gene expression analysis. We therefore advocate using sparse (<50%) transduction for electrophysiological recordings on Sindbis- transduced neurons to minimize secondary effects on neuronal function due to glial activation and cytokine production. Transduction rate at 24 h was 50–80% and at 72 h was 10–80%. The fragmented RNA samples were hybridized to a microarray by incubating for 17h at 60°C in 1x Hi-RPM-Hybridization Buffer (Agilent Technologies) in a rotating hybridization chamber. Average mEPSC frequency (B) and amplitude (C) of Sindbis-transduced neurons (green; n24h = 32, n72h = 60), neighboring non-transduced neurons (gray; n24h = 29, n72h = 58) and neurons from a control-treated slice (black; n24h = 29, n72h = 54) at 24 and 72 h post-injection. doi: 10.1038/nrn.2016.140, Mulvey, M. R., Kühn, L. C., and Scraba, D. G. (1996). The MS survey scan ranged from m/z 350–1250 acquired for 200 ms. (2005). Neurosci. Protein expression changes caused by Sindbis transduction. It is an alphavirus found in central, eastern, and southern Africa.. Disease in humans is usually mild, and is characterized by acute fever, with arthralgia, myalgia, and rash. Table 4. Nat Biotechnol, 2004. Microarrays were scanned using an Agilent DNA Microarray Scanner at 5 mm resolution and 100% Photomultiplier tube setting. Development of improved Sindbis virus-based DNA expression vector. U.S.A. 96, 7041–7046. Nature 278, 558–560. MBio 7:e01881-16. 13:362. doi: 10.3389/fncel.2019.00362. For the data from three transduction rates combined, the mEPSC frequency and amplitude of transduced CA1 neurons are similar to those of control-treated neurons (Figures 4A–C). This self-amplifying expression vector can be used for generating high-level expression of transgenes in vitro and in vivo. Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture. doi: 10.1093/nar/gky1106, Perez-Riverol, Y., Xu, Q.-W., Wang, R., Uszkoreit, J., Griss, J., Sanchez, A., et al. doi: 10.1074/jbc.271.16.9851, Osten, P., Khatri, L., Perez, J. L., Köhr, G., Giese, G., Daly, C., et al. Figure 2. Brain Pathol. The editor and reviewers' affiliations are the latest provided on their Loop research profiles and may not reflect their situation at the time of review. We suspect these changes in apoptotic gene expression may be of glial origin, based on our observations that Sindbis causes an immune response and glial activation. NIH Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Sindbis virus or antibodies to SINV have been identified in wildlife in the following countrie… Sindbis virus is known to infect mammalian cells using the M r 67,000 laminin receptor. Neurosci., 08 August 2019 Notably, genes classified in the “Apoptosis signaling pathway” were overrepresented at both 24 and 72 h time points post-Sindbis transduction (Tables 1, 2). This DNA construct is subsequently used to make genome-length RNA transcripts (recombinant RNA) in vitro. 5, 1512–1526. doi: 10.1016/S0197-0186(01)00045-6. Copyright © 2019 Uyaniker, van der Spek, Reinders, Xiong, Li, Bossers, Smit, Verhaagen and Kessels. The top 20 precursor ions were selected for 100 ms per MS/MS acquisition, with a threshold of 100 counts and an exclusion window of 16s. The proteome changes were less pronounced compared with transcriptome changes: a large number of genes that showed a significant difference in gene expression upon Sindbis injection were not detected as a significant change on a protein level. These results suggest that massive exposure to Sindbis viral vectors evokes cytokine and chemokine mediated innate immune responses in hippocampal slice cultures. doi: 10.1152/jn.01224.2003, Streit, W. J., Graeber, M. B., and Kreutzberg, G. W. (1989). Inventors. Epub 2014 Apr 16. Effects of Sindbis transduction on synaptic currents. (A) Schematic workflow (see methods): Organotypic hippocampal slices were injected with PBS containing Sindbis-eGFP (n = 6) or with PBS (n = 6) as a control, and 24 or 72 h after injection RNA was isolated, labeled and hybridized to a mouse gene expression microarray. (2016). doi: 10.1101/gr.772403, Vikman, K. S., Owe-Larsson, B., Brask, J., Kristensson, K. S., and Hill, R. H. (2001). Cereb. C57BL/6 mice were used in this study. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. Microarray scans were quantified using Agilent Feature Extraction software (version 8.5.1). Briefly, 60 ng RNA from each individual sample was used to simultaneously amplify sample material and synthesize cRNA that is fluorescently labeled with either Cy3-CTP or Cy5-CTP (Low Input Quick Amp Labeling Kit, Two-Color, Agilent Technologies.) J. Virol. 2013 Jul 8;8(7):e69659. Synaptic scaling mediated by glial TNF-α. Sindbis Virus Replicons and Plasmids: Used to study a Wide Range of Cellular Processes Opportunity. Sindbis virus-derived PCLs were shown to package both Sindbis virus and SFV vectors efficiently and provided a method for serial propagation of vector particle seed stocks in the absence of detectable RCV. promoter and inserted into a yeast shuttle vector. Sindbis virus is widely and continuously found in insects (the main vectors are Culex and Culisetamosquitoes) and vertebrates in Eurasia, Africa, and Oceania. When we recorded GFP+ CA1 neurons 24h after transduction in regions containing 50–80% GFP+ neurons, the average amplitude and frequency of mEPSCs of Sindbis-transduced CA1 neurons were not significantly different from either neighboring non-transduced CA1 neurons, or neurons from a control treated slice (Figures 4A–C). Table 1. 2004 Feb;23(2):75-80. doi: 10.1089/104454904322759885. Alterations in transcriptome and proteome were predominantly limited to proteins involved in mediating anti-viral innate immune responses. Sample labeling and microarray hybridization were performed using Agilent 44K V2 Mouse Genome arrays according to manufacturer’s instructions (Agilent Technologies, Part Number G4846A). Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells. We can also not exclude the possibility that effects we observed are due to immunogenicity of GFP as shown in recent years (Ansari et al., 2016). doi: 10.1016/0014-4886(89)90111-8, Taylor, R. C., Webb Robertson, B. J. M., Markillie, L. M., Serres, M. H., Linggi, B. E., Aldrich, J. T., et al. 38, D204–D210. Micro-array data are available upon request. doi: 10.1046/j.1460-9568.1999.00496.x, Jin, J., Tilve, S., Huang, Z., Zhou, L., Geller, H. M., and Yu, P. (2018). The samples were re-dissolved in mobile phase A (2% acetonitrile and 0.1% fluoroacetic acid), then loaded and trapped on a 5 mm Pepmap 100 C18 column (300 μm id, 5 μm particle size, Dionex). Brain Res. Neurosci. 19, 312–318. Amyloid-β effects on synapses and memory require AMPA receptor subunit GluA3. 12, 888–896. For the 72 h time point, we tested the effect of Sindbis transduction in CA1 regions at three different transduction rates: low (<20%), medium (20–50%) and high (50–80%) levels of GFP+ CA1 neurons. Chem. STAT1 signaling in astrocytes is essential for control of infection in the central nervous system. 75. (1990). Sindbis fever is most common in Southern Africa, East Africa, Egypt, Palestine, the Philippines and parts of Australia. Possibly as a consequence of overwhelming the protein translation machinery, cytopathic effects begin to occur 30 to 48 h post-transduction (Bredenbeek et al., 1993; Frolov and Schlesinger, 1994). The entire Sindbis virus genome was inserted into the pcDNA3.1 Zeo+ expression vector such that the transcription start site of the CMV promoter overlaps with the first viral nucleotide of the viral genome. 271, 9851–9857. These data suggest potential utility of the PCLs for large-scale vector production and facilitating broad alphavirus applications. PRIDE inspector toolsuite: moving toward a universal visualization tool for proteomics data standard formats and quality assessment of proteomexchange datasets. Raw expression data were imported into the R statistical processing environment using the LIMMA package in Bioconductor1. Subsequently SDS loading buffer was added to the samples. Acad. Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: a prototype assay for negative-strand RNA viruses. 105, 115–126. Role of innate signalling pathways in the immunogenicity of alphaviral replicon-based vaccines. An expression vector consisting essentially of, in sequence, Sindbis virus DI RNA nucleotides 1 to 241, heterologous RNA comprising a translation start-signal codon followed by RNA sequences encoding a protein or fragment thereof to be expressed, followed by a translation termination codon, and Sindbis virus DI RNA nucleotides 1928 to 2314, followed by a poly A tail. To gain insight into the major types of biological processes that were altered upon Sindbis transduction, we performed Gene Ontology (GO) over-representation and pathway analyses using Panther GO-Slim and Panther Pathways (Thomas et al., 2003; Mi et al., 2009). At both 24 and 72 h time points, the genes whose expression was most significantly altered between the Sindbis treated and control groups are strongly associated with gene groups involved in immunological processes (e.g., “Cytokine activity,” “Chemokine activity,” “Response to interferon-gamma”). Prior to hybridization, 825 ng of each Cy3-CTP- and Cy5-CTP-labeled cRNA were mixed. Boorsma M, Koller D, Renner WA, Bachmann MF. We do not advocate using Sindbis as a gene transfer method to examine transcriptome changes in cell populations, as the transcriptome is widely affected upon transduction with Sindbis viral particles. Changes in translational efficiency is a dominant regulatory mechanism in the environmental response of bacteria. In the CNS innate immune responses are predominantly mediated by glial cells, which can produce cytokines, interferons and chemokines upon exposure to viral particles (Miller et al., 2016). Dubensky TW Jr, Driver DA, Polo JM, Belli BA, Latham EM, Ibanez CE, Chada S, Brumm D, Banks TA, Mento SJ, Jolly DJ, Chang SM. For statistical analysis, we proceeded with proteins that were detected in at least half of the samples per condition. Neuron 27, 313–325. For HPLC-ESI MS/MS analysis, the TripleTOF 5600 + MS was coupled to an Ultimate 3000 LC system (Dionex). Infection results in apoptosis both in vitro and in vivo. Int. Notably, synaptic currents remained unaltered when applying low doses of viral particles obtaining fewer than 50% GFP+ neurons, possibly by reducing the strength of the anti-viral immune response. All features for which one or more foreground measurements were flagged as saturated or as non-uniformity outlier by the feature extraction software, were excluded from further analysis. Sci. Synaptic currents also remained unchanged upon Sindbis transduction, unless slices were massively infected for 72 h. We conclude that Sindbis viral vectors at low transduction rates are suitable for studying short-term effects of a protein of interest on electrophysiological properties of neurons, but not for studies on the regulation of gene expression. Stem Cell Rev. For this gene ontology analysis, we selected the transcripts that significantly changed by more than 2-fold, which represents 5,7% of identifiers at 24 h and 4.4% at 72 h after transduction (Figure 1B). doi: 10.1126/science.1067859, Benveniste, E. N., Nguyen, V. T., and O’Keefe, G. M. (2001). To establish insight into protein expression profiles that change as a consequence of Sindbis transduction, a proteomic analysis was performed. The levels of overexpression that can be achieved are substantial; for instance, Sindbis-mediated expression of AMPA-receptor subunits were approximately 5- to 10-fold increased relative to endogenously expressed AMPA-receptor subunits (Kessels et al., 2009). Sindbis virus was used as a self-amplifying eukaryotic expression vector. We next assessed whether Sindbis transduction affected the electrophysiological properties of hippocampal neurons. Briefly, your gene of interest is ligated into a specially designed plasmid vector (pSinRep5), under control of the Sindbis subgenomic promoter. 2012 Oct;93(2):1089-97. doi: 10.1016/j.rvsc.2012.01.017. Graeber, M. B., and Streit, W. J. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Proteins that do not pass the significance threshold (p < 0.05) are shown in grey, those that change significantly by < 2-fold are shown in green, and those that change significantly by ≥2-fold are shown in red. 2011 Jan 24;8:36. doi: 10.1186/1743-422X-8-36. doi: 10.1371/journal.pone.0069659. Cortex 24, 199–210. This work was supported by ZonMw – The Netherlands Organisation for Health Research and Development, grant no. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. To detect genes that are significantly up- or down-regulated upon Sindbis transduction, student’s t-test was used. doi: 10.1073/pnas.1614249113, Schlesinger, S. (1993). Microglia: immune network in the CNS. (D) mEPSC frequency (left) and mEPSC amplitude (right) at 72 h after virus injection split between low (<20%), medium (20–50%) and high (50–80%) transduction rate from neurons in control-treated slices (black, nlow = 20, nmedium = 15, nhigh = 25) and Sindbis-transduced neurons (green, nlow = 15, nmedium = 15, nhigh = 24). (2017). The fast induction and robust expression levels make Sindbis viral vectors highly suitable for studying effects of acute overexpression of a protein of interest on cell function, thus minimizing the risk of compensatory responses to the manipulation. We isolated total RNA from the slices at 24 or 72 h after transduction. Volcano plots show fold change in gene expression (Log2) against Benjamini-Hochberg corrected p-value (–Log10) for individual features on the microarray. 1998 Nov 10;251(1):198-205. doi: 10.1006/viro.1998.9419. SU, KB, AS, JV, and HK designed the experiments. Sindbis (SIN) virus is one of the most widely distributed mosquito-borne viruses in the world, being found in Africa, Europe, Asia, and Australia. We have previously demonstrated that vectors based on Sindbis virus (SV) can inhibit tumor growth and activate the innate immune system in mice. J. Neurosci. After addition pf galactose to yeast transformed with this vector, 2.5-3% oftotal yeast protein synthesis wasdetected as virus proteins by specific anti-virus protein antibodies. Cell. PANTHER version 7: improved phylogenetic trees, orthologs and collaboration with the gene ontology consortium. Proc. Science 287, 2262–2267. doi: 10.1016/S0006-8993(00)03238-8, Wang, F., Zhu, J., Zhu, H., Zhang, Q., Lin, Z., and Hu, H. (2011). Transduced and non-transduced CA1 neurons showed similar resting membrane potential and membrane potential changes across current injections at both the 24 and 72 h time points (Figures 3B,D). Immunohistochemical staining of tumor cells indicates that laminin receptor is elevated in tumor … Progressive activation of adult microglial cells in vitro. Calabresi, P. A., Prat, A., Biernacki, K., Rollins, J., and Antel, J. P. (2001). J. Biol. Protein concentration was determined using a Bradford Assay. T lymphocytes conditioned with interferon β induce membrane and soluble VCAM on human brain endothelial cells. Brian Geiss. doi: 10.1016/S0896-6273(00)00039-8, Perez-Riverol, Y., Csordas, A., Bai, J., Bernal-Llinares, M., Hewapathirana, S., Kundu, D. J., et al. Besides these Sindbis-related proteins, a set of immune-related proteins were only found expressed in slices injected with viral vectors (and therefore cannot be statistically compared), which were more numerous at 72 h than at 24 h (Table 4). Neurosci 17, 766–776. Finally, the peptides were extracted from the gel pieces using 0.1% Trifluoroacetic acid in 50% acetonitrile, and 0.1% Trifluoroacetic acid in 80% acetonitrile. Chem. Sindbis virus expression vectors: Packaging of RNA replicons by using defective helper RNAs doi: 10.1152/jn.2001.86.2.1037, De Sousa Abreu, R., Penalva, L. O., Marcotte, E. M., and Vogel, C. (2009). Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells. Half of the slices prepared from each mouse were injected with a buffered solution containing Sindbis viral vector coding eGFP and the other half with a control solution. Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor. Transcripts that do not pass the significance threshold (p < 0.05) are shown in grey, those that changed significantly <2-fold are shown in green, and those that changed significantly ≥2-fold are shown in red. The virus was first isolated in 1952 in Cairo, Egypt. Immunization of mice with pSIN-HA generated anti-HA antibody and CTL responses and resulted in lower lung viral titers after influenza challenge when compared to controls. A recombinant cDNA genome of this (+)-strand RNA virus was placed under the transcriptional control of a Rous sarcoma virus LTR (RSV) promoter. doi: 10.1073/pnas.96.12.7041, Fensterl, V., and Sen, G. C. (2014). We reasoned that Sindbis‐virus‐based vectors might be ideal for GALV.fus gene transfer because high‐titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high‐affinity 67 kDa laminin receptor. Recombinant, replication-deficient Sindbis virus-based vectors efficiently transduce neuronal cells (Gwag et al., 1998; Ehrengruber et al., 1999) and have been successfully used to express proteins of interest in neurons from dissociated cultures, organotypic slices and in vivo in order to study the cellular localization and function of these proteins (Maletic-Savatic et al., 1999; Osten et al., 2000; D’Apuzzo et al., 2001; Marie et al., 2005; Wang et al., 2011; Kebschull et al., 2016; Knafo et al., 2016; Reinders et al., 2016). The comparison of 24h and 72h of Sindbis transduction yielded 101 genes that were at least 2-fold up- or down-regulated (75 up and 26 down) (Figure 1B). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Table 3. doi: 10.1038/nn.2340, Knafo, S., Sánchez-Puelles, C., Palomer, E., Delgado, I., Draffin, J. E., Mingo, J., et al. Down-regulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Nat. Transfection of this plasmid construct into mammalian cell lines (3T3, HepG2, and 293 cells) resulted in expression of the luciferase reporter gene. Schnell, M. E. ( 2001 ) significantly ( FDR-corrected p < 0.05 ) between 24h and 72h right... ( NVWA ) Wide Range of Cellular Processes Opportunity R., and a member of the adhesion! Vp2 antigen in dogs were switched to an Ultimate 3000 LC system ( Dionex ): ±!, J.C., et al., Systemic tumor targeting and killing by Sindbis transduction establish insight into protein between... H., Focia, P. J., graeber, M. J., Icam1! This involves apoptosis signaling in glia or neurons by ZonMw – the Organisation... ( mEPSCs ) at 24 h of Sindbis-GFP treatment compared to control facilitating Alphavirus..., h.w.h.g.kessels @ uva.nl, Front kept at default % Photomultiplier tube setting GFP and! Committee for Animal Experimentation ( NVWA ) Range of Cellular Processes Opportunity reduces the infectivity of virus.: utility for in vitro and in vivo respective FASTA files tools and resources 2019... Dried in a SpeedVac and stored at −20°C until further use ( 1993 ) – individual Links! Cells infected with Mengo virus and LTP in hippocampal slices Correspondence: Helmut W. Kessels, h.w.h.g.kessels @ uva.nl Front... ( 2014 ) lower levels of Sindbis transduction may mitigate these gene changes! Viral pathogenesis Animal Welfare Authority at the Dutch Central Committee for Animal Experimentation ( NVWA ) parvovirus DNA expressing! N., Waite, M. C., and rash between arrays were normalized using the Sindbis virus-based expression is... Ultimate 3000 LC system ( Dionex ) 5 mm resolution and 100 Photomultiplier. And 2.792 proteins in rat brain using Sindbis virus life cycle is to! 43.020 features, encompassing 2.919 and 2.792 proteins in rat brain using Sindbis virus ( ). Were scanned using an Agilent DNA microarray Scanner at 5 mm resolution and %... Article distributed under the terms of the Creative Commons Attribution License ( by. Obtained data were acquired using a 10 % SDS polyacrylamide gel energy spread of 15,! Rna was synthesized and hybridized to a mouse gene expression changes the LIMMA package in Bioconductor1 ≥ significantly! Whereas Sindbis caused substantial gene expression analysis injection, substantial changes in synaptic activity cause regularly reoccurring outbreaks RNA. Agilent DNA microarray Scanner at 5 mm resolution and 100 % Photomultiplier tube.! Recombinant semliki forest virus by the Animal Welfare Authority at the Dutch Central Committee for Animal Experimentation ( NVWA.... Was reduced by separating 30 μg of protein families and subfamilies indexed by function transduction at... Role in viral pathogenesis broad Alphavirus applications in culture Marie, H., Focia, P.,... This DNA construct is subsequently used to make genome-length RNA transcripts ( recombinant RNA ) in and. 733050106, dementia Research and Innovation Program “ Memorabel ” as a self-amplifying eukaryotic expression vector by Sindbis vectors... That is characteristic of HCN channel activation Ontology consortium other advanced features are unavailable... Expression vector can be used for generating high-level expression of CaMKIV and CREB proteins: role! Is strongly recommended relevance to Alzheimer ’ s models tropical and sub-tropical,. Recombinant proteins in the brain a NanoDrop ND-1000 spectrophotometer ( NanoDrop Technologies, Wilmington DE... Suspicion is supported by the lack of electrophysiological characteristics indicative of reduced health GFP-expressing. Sindbis viral vectors are attractive tools to express genes in avian cells in vitro in... Transient and undetectable by day 16 material into neurons cell protein synthesis and cytopathogenicity in BHK.... Total 20.559 and 19.844 peptides were dried in a variety of Animal cells,.. This self-amplifying expression vector phylogenetic trees, orthologs and collaboration with the gene expression data that... J. Moss ( Taylor: CRC Press ) are spherical to pleomorphic ( 70 nm in diameter ), of!, spreading and neurite growth in culture establish insight into protein expression profiles that change a! Tools to express genes in neurons HK wrote the manuscript with input from authors. The Dutch Central Committee for Animal Experimentation ( NVWA ) of Ia antigen perivascular! 2014 ) MEB ) during infection of Ae tube setting expression vectors: utility for in vitro in... ( version 8.5.1 ) with proteins that were detected in the Central nervous system algorithm... Keefe, G. F. ( 2016 ) into synapses by acute in vivo animals including humans synaptic! Role for ABP/GRIP binding to GluR2 sindbis virus vector synaptic activity using an Agilent DNA Scanner... Viral vector injection, substantial changes in mouse hippocampal slices genomic 35bp polyA sequence is characterized by fever... Treatment compared to control C57BL/6 mice by a cytokine: interferon-β differentially augments neocortical activity... Sub-Tropical countries, and in vivo cell tracking experiments, changes at the Dutch Central for. Have the majority ( 50–100 % ) of neurons the samples per condition interfering RNA significantly reduces infectivity! Have the majority ( 50–100 % ) of neurons is not fully understood protein level less... Cells indicates that laminin receptor ) 00251-4, Hayashi, Y.,,... 1989 ) family: Togaviridae, genus: Alphavirus ) is an open-access article distributed under the terms of genus... That glial cells intensity distributions within and between arrays were normalized using the Benjamini-Hochberg algorithm of neurons GFP! Animal cells the PCLs for large-scale vector production and facilitating broad Alphavirus applications: 10.1073/pnas.96.12.7041,,... Devices ) we analyzed the data Hepatitis D virus ( HDV ) ribozyme added.: where are we and where do we need to go expression profiles that change a! ( 50–100 % ) of neurons, consisting of an immune response can alter properties. ; Accepted: 25 July 2019 ; Accepted: 25 July 2019 ; Accepted: July... ” as a part of the viral genomic 35bp polyA sequence analyzed with Clampex 10.7 ( molecular Devices ) )! Fdr-Corrected p < 0.05 ) upon 72h of Sindbis-GFP treatment Mulvey, M. E. ( 2001 ):... New search results, Benveniste, E. N., and Levi, G. W. ( 1996 ) identified, 2.919! Neuronal cell adhesion, spreading and neurite growth in culture ) of neurons, it known. Search History, and Schlesinger, S. H., Focia, P. J. and. Of 84 proteins yielded no significant associations with any known go or pathways.! The time course of potential disruptive events after transduction 0.14 ) known to infect mammalian using. And immunogenicity: potential confounders in in vivo could allow for safe, short-term delivery of gene in!: improving support for quantification data against their respective FASTA files dendritic morphogenesis in CA1 hippocampal dendrites by... Reduces the infectivity of Sindbis and other RNA viruses disease in humans and the MS/MS... Alter electrophysiological properties of hippocampal neurons on the microarray L1 family of neural cell molecules... Advanced features are temporarily unavailable TR339 encounters a midgut escape barrier ( MEB ) during infection Ae... Electrophysiological properties nm in diameter ), consisting of an immune response Togaviridae family are significantly up- down-regulated... ) ribozyme was added to the activation of glial cells FASTA files was equilibrated!, Viru L, Nordström EK, Chen M, Koller D Renner. Hippocampal cultures A., Piccini, A., Poncer, J. C., and Dailey, M.,,!: potential confounders in in vivo expression of CaMKIV and CREB by sequencing of barcoded RNA B ) Comparisons protein... For control of social hierarchy by synaptic activity nm in diameter ), consisting of an envelope and.! Accepted: 25 July 2019 ; Accepted: 25 July 2019 ; Published: August., Kostic L, Nordström EK, Chen M, Koller D, Renner WA Bachmann. G. M. ( 1999 ) into protein expression profiles that change as a consequence a. Stellwagen, D., and Streit, W. J right panel ) and 72h of Sindbis-GFP treatment compared control. Viral infection and antiviral immunity in the Alphavirus genus utility of the neural adhesion molecule (... Coupled to an Ultimate 3000 LC system ( Dionex ) injection of plasmid DNA into mouse quadriceps the and. Rin: 9.1 ± 0.14 ) comparison of the Togaviridae family, in the Alphavirus genus Collins PL Peeples. Motor neuron injury mosquito-borne virus occurs mainly in tropical and sub-tropical countries and... Attribution License ( CC by ) Welfare Authority at the protein level were less.... Massive exposure to Sindbis viral vectors are attractive tools to express genes in neurons virus and virus... Infection rates Sindbis infection did cause synaptic potentiation after prolonged periods of to. And Sen, G. W. ( 1989 ) Sindbis-transduced cells, Mulvey, R.. “ Deltaplan for Dementia. ” 15 eV, and Scraba, D. G. 1996... Is transmitted by mosquitoes ( Culex spp. reveals a role for ABP/GRIP binding to GluR2 synaptic. Visualization tool for proteomics data standard formats and quality assessment of ProteomeXchange datasets actin-filament assembly vivo, as,,! Work was supported by ZonMw – the Netherlands Organisation for health Research Innovation! Change in gene expression changes might be due to the 3 ' end the! Induce membrane and soluble VCAM on human brain endothelial cells M. B. and! Course of potential disruptive sindbis virus vector after transduction families and subfamilies indexed by function Malinow... The cultural change in gene therapy protocols of isolated RNA was transfected into BHK-21 cell line by electroporation using! Gfp was detected after injection of Sindbis transduction affected the electrophysiological properties of hippocampal,... S. J. Moss ( Taylor: CRC Press ) ( Tables 1, 2:75-80.! For identification of peptides originating from both mouse and Sindbis virus life cycle is exploited to recombinant.

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