scholarly journals Discovery of a prevalent picornavirus by visualizing zebrafish immune responses

2019 ◽  
Author(s):  
Keir M. Balla ◽  
Marlen C. Rice ◽  
James A. Gagnon ◽  
Nels C. Elde
Keyword(s):  
Fluorescent Protein ◽  
Gene Promoter ◽  
Immune Defense ◽  
Virus Infections ◽  
Sequencing Data ◽  
Public Data ◽  
Picornavirus Infection ◽  
Vertebrate Hosts ◽  
Green Fluorescent ◽  
New Strategies

AbstractThe discovery of new viruses currently outpaces our capacity for experimental examination of infection biology. To better couple virus discovery with immunology, we genetically modified zebrafish to visually report on virus infections. After generating a strain that expresses green fluorescent protein (GFP) under an interferon-stimulated gene promoter, we repeatedly observed transgenic larvae spontaneously expressing GFP days after hatching. RNA sequencing of GFP-positive zebrafish revealed a picornavirus distantly related to known viruses. We detected this virus in publicly available sequencing data from seemingly asymptomatic zebrafish in research institutes world-wide. These data revealed widespread tissue tropism and identified the clonal CG2 strain as notably vulnerable to picornavirus infection. Infection induces the expression of numerous antiviral immune defense genes in zebrafish, an effect also evident in public data. Our study describes a prevalent picornavirus that infects zebrafish, and provides new strategies for simultaneously discovering viruses and their impact on vertebrate hosts.

scholarly journals The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium

2001 ◽  
Vol 67 (2) ◽  
pp. 948-955 ◽  
Author(s):  
Biao Ma ◽  
Mary B. Mayfield ◽  
Michael H. Gold
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Phanerochaete Chrysosporium ◽  
Fluorescent Protein ◽  
Schizophyllum Commune ◽  
Extracellular Fluid ◽  
Gene Promoter ◽  
Green Fluorescent Protein Gene ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfpcoding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of theegfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly withegfp, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of theegfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of theegfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studyingcis-acting elements in the mnp1 gene promoter.

FC26-06 - Development of interneurons expressing serotonin 3A subtype receptor and sleep regulation

2011 ◽  
Vol 26 (S2) ◽  
pp. 1964-1964
Author(s):  
K. Vucurovic ◽  
C. Béra-Potelle ◽  
O. Andrei ◽  
A. Kaladjian ◽  
T. Vitalis
Keyword(s):  
Fluorescent Protein ◽  
Gene Promoter ◽  
In Utero ◽  
Morphological Properties ◽  
Sleep Regulation ◽  
Adult Age ◽  
In Utero Transplantation ◽  
Transgenic Embryo ◽  
Developmental Characteristics ◽  
Green Fluorescent

IntroductionSerotonin (5-HT) is known to play a key role in a number of psychiatric disorders. The effects of abnormal 5-HT neurotransmission on the modulation of circadian rhythm have been proposed to be one of the mechanisms underlying these disorders.Objectives and aimsHere we describe developmental characteristics of interneurones (IN) expressing 5-HT type 3A receptor (5-HT3AR) in mice. This receptor is a ligand-gated ion channel which activation leads to a rapid excitatory response in neurons.The expression of 5-HT3AR during embryonic development in mice is mainly restricted to caudal ganglionic eminence (CGE) and entopedoncular area (EPA). We hypothesized that these regions are those specifically involved in the neurogenesis of the IN expressing 5-HT3AR.MethodsTo assess this hypothesis, we used homochronic in utero grafts. Cells were collected from CGE and EPA regions of transgenic mice embryos expressing Green Fluorescent Protein under the control of 5-HT3AR gene promoter. After in utero transplantation in non transgenic embryo, we examined at adult age the location of the grafted cells, as well as their morphological properties.ResultsWe observed that CGE-derived cells gave rise to IN that mostly populated the neocortex and the hippocampus, whereas EPA-derived cells integrated the amygdala, the pyriform cortex and the Ventro-Lateral Pre-Optic (VLPO) nucleus. Moreover, the VLPO EPA-derived cells are similar to “sleep active cells” found in this region and responsible of sleep induction.ConclusionThese results lead to the better understanding of the IN expressing 5HT3AR development. Its activation is possibly implicated in triggering sleep mechanismes.

scholarly journals Pituitary Corticotroph Ontogeny and Regulation in Transgenic Zebrafish

2003 ◽  
Vol 17 (5) ◽  
pp. 959-966 ◽  
Author(s):  
Ning-Ai Liu ◽  
Haigen Huang ◽  
Zhongan Yang ◽  
Wiebke Herzog ◽  
Matthias Hammerschmidt ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cell Mass ◽  
Gene Promoter ◽  
Genetic System ◽  
Regulatory Elements ◽  
Transgenic Zebrafish ◽  
Posterior Pituitary ◽  
Gfp Expression ◽  
Green Fluorescent

Abstract We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18–20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and αMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10−5m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.

A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport

2012 ◽  
Vol 303 (6) ◽  
pp. F886-F892 ◽  
Author(s):  
Nicolas Markadieu ◽  
Pedro San-Cristobal ◽  
Anil V. Nair ◽  
Sjoerd Verkaart ◽  
Ellen Lenssen ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Fluorescent Protein ◽  
Cell Model ◽  
Gene Promoter ◽  
Complex Object ◽  
Concentration Gradients ◽  
Nacl Transport ◽  
Cell Monolayers ◽  
Green Fluorescent ◽  
First Time

Studying the molecular regulation of the thiazide-sensitive Na+-Cl− cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na+ transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm2. The monolayers also established opposing transcellular concentration gradients of Na+ and K+. Radioactive 22Na+ flux experiments showed a net apical-to-basolateral thiazide-sensitive Na+ transport across the monolayers. Both hypotonic low-chloride medium and 1 μM angiotensin II increased this 22Na+ transport significantly by four times, which could be totally blocked by 100 μM hydrochlorothiazide. Angiotensin II-stimulated 22Na+ transport was also inhibited by 1 μM losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na+-Cl− transport.

scholarly journals SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

2017 ◽  
Vol 38 (2) ◽  
Author(s):  
Venkatesh Kota ◽  
Gunhild Sommer ◽  
E. Starr Hazard ◽  
Gary Hardiman ◽  
Jeffery L. Twiss ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Hek293 Cells ◽  
Sequencing Data ◽  
Protein Levels ◽  
Specific Mrnas ◽  
Green Fluorescent

ABSTRACTThe cancer-associated RNA-binding protein La is posttranslationally modified by phosphorylation and sumoylation. Sumoylation of La regulates not only the trafficking of La in neuronal axons but also its association with specific mRNAs. Depletion of La in various types of cancer cell lines impairs cell proliferation; however, the molecular mechanism whereby La supports cell proliferation is not clearly understood. In this study, we address the question of whether sumoylation of La contributes to cell proliferation of HEK293 cells. We show that HEK293 cells stably expressing green fluorescent protein (GFP)-tagged wild-type La (GFP-LaWT) grow faster than cells expressing a sumoylation-deficient mutant La (GFP-LaSD), suggesting a proproliferative function of La in HEK293 cells. Further, we found that STAT3 protein levels were reduced in GFP-LaSDcells due to an increase in STAT3 ubiquitination and that overexpression of STAT3 partially restored cell proliferation. Finally, we present RNA sequencing data from RNA immunoprecipitations (RIPs) and report that mRNAs associated with the cell cycle and ubiquitination are preferentially bound by GFP-LaWTand are less enriched in GFP-LaSDRIPs. Taken together, results of our study support a novel mechanism whereby sumoylation of La promotes cell proliferation by averting ubiquitination-mediated degradation of the STAT3 protein.

scholarly journals Green Fluorescent Protein as a Marker in Plasmodium berghei Transformation

1999 ◽  
Vol 67 (5) ◽  
pp. 2602-2606 ◽  
Author(s):  
Ali A. Sultan ◽  
Vandana Thathy ◽  
Victor Nussenzweig ◽  
Robert Ménard
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Plasmodium Berghei ◽  
Fluorescent Protein ◽  
Single Copy ◽  
Malarial Parasite ◽  
Molecular Studies ◽  
Direct Fluorescence ◽  
Vertebrate Hosts ◽  
Green Fluorescent

ABSTRACT We present a new marker that confers both resistance to pyrimethamine and green fluorescent protein-based fluorescence on the malarial parasite Plasmodium berghei. A single copy of the cassette integrated into the genome is sufficient to direct fluorescence in parasites throughout the life cycle, in both its mosquito and vertebrate hosts. Erythrocyte stages of the parasite that express the marker can be sorted from control parasites by flow cytometry. Pyrimethamine pressure is not necessary for maintaining the cassette in transformed parasites during their sporogonic cycle in mosquitoes, including when it is borne by a plasmid. This tool should thus prove useful in molecular studies of P. berghei, both for generating parasite variants and monitoring their behavior.

scholarly journals Histone H1 Is Required for Proper Regulation of Pyruvate Decarboxylase Gene Expression in Neurosporacrassa

2003 ◽  
Vol 2 (2) ◽  
pp. 341-350 ◽  
Author(s):  
H. Diego Folco ◽  
Michael Freitag ◽  
Ana Ramón ◽  
Esteban D. Temporini ◽  
María E. Alvarez ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Carbon Sources ◽  
Pyruvate Decarboxylase ◽  
Chimeric Protein ◽  
Gene Promoter ◽  
Histone H1 ◽  
Globular Domain ◽  
Mutagen Sensitivity ◽  
Green Fluorescent

ABSTRACT We show that Neurospora crassa has a single histone H1 gene, hH1, which encodes a typical linker histone with highly basic N- and C-terminal tails and a central globular domain. A green fluorescent protein-tagged histone H1 chimeric protein was localized exclusively to nuclei. Mutation of hH1 by repeat-induced point mutation (RIP) did not result in detectable defects in morphology, DNA methylation, mutagen sensitivity, DNA repair, fertility, RIP, chromosome pairing, or chromosome segregation. Nevertheless, hH1 mutants had mycelial elongation rates that were lower than normal on all tested carbon sources. This slow linear growth phenotype, however, was less evident on medium containing ethanol. The pyruvate decarboxylase gene, cfp, was abnormally derepressed in hH1 mutants on ethanol-containing medium. This derepression was also found when an ectopically integrated fusion of the cfp gene promoter to the reporter gene hph was analyzed. Thus, Neurospora histone H1 is required for the proper regulation of cfp, a gene with a key role in the respiratory-fermentative pathway.

scholarly journals Generation of Transgenic Rats Expressing Green Fluorescent Protein in S-100β-Producing Pituitary Folliculo-Stellate Cells and Brain Astrocytes

Endocrinology ◽  
2007 ◽  
Vol 148 (4) ◽  
pp. 1518-1523 ◽  
Author(s):  
Eisuke Itakura ◽  
Kousuke Odaira ◽  
Kotaro Yokoyama ◽  
Marumi Osuna ◽  
Takahiko Hara ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gene Promoter ◽  
Anterior Lobe ◽  
Transgenic Rats ◽  
Specific Expression ◽  
Sustentacular Cells ◽  
Intron 1 ◽  
S 100 ◽  
Green Fluorescent

Folliculo-stellate (FS) cells are known to act as sustentacular cells or scavenger cells in the anterior lobe. However, the precise function and origin of FS cells are still under discussion. Like brain astrocytes, FS cells contain S-100β protein, and FS cells can be detected immunocytochemically using antibodies for S-100β protein after fixation; however, living FS cells can not be detected. The generation of transgenic rats expressing green fluorescent protein (GFP) under the control of S-100β protein gene promoter may allow the detection of living FS cells, which may be an excellent tool for the study of FS cells. With the aim of generation of transgenic rats, we analyzed the promoter activity of the S-100β gene and found that intron 1 is important for cell-specific expression of the S-100β gene. Therefore, we obtained a DNA construct containing GFP gene under a part of the S-100 promoter with intron 1. We transfected the construct into rat embryos and succeeded in generating transgenic rats. The transgenic rats expressed GFP in FS cells specifically in the anterior lobe. GFP is also expressed in other known S-100β-expressing cells, i.e. brain astrocytes, adipocytes, and chondrocytes. We believe that the newly generated transgenic rats will provide a new approach for the study of FS cells and other S-100β protein-producing cells.

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