A Modified Sindbis Vector for Prolonged Gene Expression in Neurons

2003 ◽  
Vol 90 (4) ◽  
pp. 2741-2745 ◽  
Author(s):  
Andreas Jeromin ◽  
Li-Lian Yuan ◽  
Andreas Frick ◽  
Paul Pfaffinger ◽  
Daniel Johnston
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Time Window ◽  
Essential Gene ◽  
Functional Expression ◽  
Host Protein ◽  
Neuronal Structure ◽  
Voltage Gated Ion Channels ◽  
Green Fluorescent

Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for ≥6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.

NRSF Causes cAMP-Sensitive Suppression of Sodium Current in Cultured Hippocampal Neurons

2002 ◽  
Vol 88 (1) ◽  
pp. 409-421 ◽  
Author(s):  
H. Nadeau ◽  
H. A. Lester
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Sodium Current ◽  
Striking Similarity ◽  
Secondary Effects ◽  
Functional Effect ◽  
Green Fluorescent ◽  
Altered Sensitivity ◽  
Cultured Hippocampal Neurons

The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na+channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1–2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na+channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K+currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic “silencer” and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

Abundant GFP Expression and LTP in Hippocampal Acute Slices by In Vivo Injection of Sindbis Virus

2001 ◽  
Vol 86 (2) ◽  
pp. 1037-1042 ◽  
Author(s):  
Massimo D'Apuzzo ◽  
Georgia Mandolesi ◽  
Gerald Reis ◽  
Erin M. Schuman
Keyword(s):  
Hippocampal Neurons ◽  
Sindbis Virus ◽  
Fluorescent Protein ◽  
Hippocampal Slices ◽  
Pyramidal Cells ◽  
Long Term Potentiation ◽  
Neuronal Structure ◽  
Adult Rats ◽  
Acute Hippocampal Slices

Virus-mediated gene transfer into neurons is a powerful tool for the analysis of neuronal structure and function. Recombinant sindbis virus has been previously used to study protein function in hippocampal neuron cultures as well as in hippocampal organotypic slice cultures. Nevertheless, some concern still exists about the physiological relevance of these cultured preparations. Acute hippocampal slices are a widely used preparation for the study of synaptic transmission, but currently recombinant gene delivery is usually achieved only through time-consuming transgenic techniques. In this study, we show that a subregion of the CA1 area in acute hippocampal slices can be specifically altered to express a gene of interest. A sindbis virus vector carrying an enhanced green fluorescent protein (EGFP) reporter was injected in vivo into the hippocampus of adult rats. After 18 h, rats were killed, and acute hippocampal slices, infected in the CA1 field, were analyzed morphologically and electrophysiologically. Infected slices showed healthy and stable electrophysiological responses as well as long-term potentiation. In addition, infected pyramidal cells were readily recognized in living slices by two-photon imaging. Specifically, the introduction of an EGFP-Actin fusion protein greatly enhanced the detection of fine processes and dendritic spines. We propose this technique as an efficient tool for studying gene function in adult hippocampal neurons.

scholarly journals Short Tetracysteine Tags to β-Tubulin Demonstrate the Significance of Small Labels for Live Cell Imaging

2004 ◽  
Vol 15 (12) ◽  
pp. 5616-5622 ◽  
Author(s):  
Martin Andresen ◽  
Rita Schmitz-Salue ◽  
Stefan Jakobs
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Open Reading Frames ◽  
Host Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent ◽  
Β Tubulin

Genetically encoded tags are of fundamental importance for live cell imaging. We show that small tetracysteine (TetCys) tags can be highly advantageous for the functionality of the host protein compared with large fluorescent protein tags. One to three concatenated small TetCys tags as well as the large green fluorescent protein (GFP) were fused by integrative epitope tagging to the C terminus of β-tubulin (Tub2) in the budding yeast Saccharomyces cerevisiae. The increasing tag size correlated with functional interference to the host protein. Tub2 tagged with either 1×TetCys (10 amino acids [aa]) or 2×TetCys (20 aa) was able to substitute Tub2 in haploid cells. In contrast, C-terminal tagging of Tub2 with 3×TetCys (29 aa) or with GFP (244 aa) resulted in nonviable haploid cells. Cells expressing Tub2-1×TetCys or Tub2-2×TetCys were stained with FlAsH, which selectively binds to the TetCys-tag. The stained cells displayed dynamic FlAsH-labeled microtubules and low cellular background fluorescence. The presented approach to tag open reading frames (ORFs) at their native loci with very small TetCys-tags and the subsequent visualization of the tagged proteins in vivo can be extended in principle to any ORF in S. cerevisiae.

scholarly journals Spike-timing-dependent BDNF secretion and synaptic plasticity

2014 ◽  
Vol 369 (1633) ◽  
pp. 20130132 ◽  
Author(s):  
Hui Lu ◽  
Hyungju Park ◽  
Mu-Ming Poo
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Time Window ◽  
Hippocampal Slices ◽  
Long Term Potentiation ◽  
Spike Timing ◽  
Dependent Manner ◽  
Induced Responses ◽  
Before And After ◽  
Green Fluorescent

In acute hippocampal slices, we found that the presence of extracellular brain-derived neurotrophic factor (BDNF) is essential for the induction of spike-timing-dependent long-term potentiation (tLTP). To determine whether BDNF could be secreted from postsynaptic dendrites in a spike-timing-dependent manner, we used a reduced system of dissociated hippocampal neurons in culture. Repetitive pairing of iontophoretically applied glutamate pulses at the dendrite with neuronal spikes could induce persistent alterations of glutamate-induced responses at the same dendritic site in a manner that mimics spike-timing-dependent plasticity (STDP)—the glutamate-induced responses were potentiated and depressed when the glutamate pulses were applied 20 ms before and after neuronal spiking, respectively. By monitoring changes in the green fluorescent protein (GFP) fluorescence at the dendrite of hippocampal neurons expressing GFP-tagged BDNF, we found that pairing of iontophoretic glutamate pulses with neuronal spiking resulted in BDNF secretion from the dendrite at the iontophoretic site only when the glutamate pulses were applied within a time window of approximately 40 ms prior to neuronal spiking, consistent with the timing requirement of synaptic potentiation via STDP. Thus, BDNF is required for tLTP and BDNF secretion could be triggered in a spike-timing-dependent manner from the postsynaptic dendrite.

scholarly journals A Conserved Mechanism of Synaptogyrin Localization

2001 ◽  
Vol 12 (8) ◽  
pp. 2275-2289 ◽  
Author(s):  
Hongjuan Zhao ◽  
Michael L. Nonet
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
C Elegans ◽  
C Terminus ◽  
Green Fluorescent ◽  
Cultured Hippocampal Neurons

We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-taggedCaenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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