scholarly journals Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function

BioTechniques ◽  
2008 ◽  
Vol 45 (6) ◽  
pp. 659-668 ◽  
Author(s):  
Richard J. Stanton ◽  
Brian P. McSharry ◽  
Melanie Armstrong ◽  
Peter Tomasec ◽  
Gavin W.G. Wilkinson
Keyword(s):  
High Throughput ◽  
Gene Function ◽  
Adenovirus Vector ◽  
Vector Systems

scholarly journals High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: Alternative expression modes and gene function correlates

2010 ◽  
Vol 20 (5) ◽  
pp. 636-645 ◽  
Author(s):  
J. Matsumoto ◽  
K. Dewar ◽  
J. Wasserscheid ◽  
G. B. Wiley ◽  
S. L. Macmil ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
High Throughput ◽  
Gene Function ◽  
Ciona Intestinalis ◽  
Alternative Expression

scholarly journals Toward the Development of a Virus-Cell-Based Assay for the Discovery of Novel Compounds against Human Immunodeficiency Virus Type 1

2003 ◽  
Vol 47 (2) ◽  
pp. 501-508 ◽  
Author(s):  
Martin E. Adelson ◽  
Annmarie L. Pacchia ◽  
Malvika Kaul ◽  
Robert F. Rando ◽  
Yacov Ron ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Discovery ◽  
Human Immunodeficiency Virus Type ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cycle ◽  
Vector Systems ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The emergence of human immunodeficiency virus type 1 (HIV-1) strains resistant to highly active antiretroviral therapy necessitates continued drug discovery for the treatment of HIV-1 infection. Most current drug discovery strategies focus upon a single aspect of HIV-1 replication. A virus-cell-based assay, which can be adapted to high-throughput screening, would allow the screening of multiple targets simultaneously. HIV-1-based vector systems mimic the HIV-1 life cycle without yielding replication-competent virus, making them potentially important tools for the development of safe, wide-ranging, rapid, and cost-effective assays amenable to high-throughput screening. Since replication of vector virus is typically restricted to a single cycle, a crucial question is whether such an assay provides the needed sensitivity to detect potential HIV-1 inhibitors. With a stable, inducible vector virus-producing cell line, the inhibitory effects of four reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, and didanosine) and one protease inhibitor (indinavir) were assessed. It was found that HIV-1 vector virus titer was inhibited in a single cycle of replication up to 300-fold without affecting cell viability, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. Thus, it seems likely that HIV-1-derived vector systems can be utilized in a novel fashion to facilitate the development of a safe, efficient method for screening compound libraries for anti-HIV-1 activity.

scholarly journals A High-Throughput Gene-Silencing System for the Functional Assessment of Defense-Related Genes in Barley Epidermal Cells

2005 ◽  
Vol 18 (8) ◽  
pp. 755-761 ◽  
Author(s):  
D. Douchkov ◽  
D. Nowara ◽  
U. Zierold ◽  
P. Schweizer
Keyword(s):  
Gene Silencing ◽  
High Throughput ◽  
Gene Function ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Epidermal Cells ◽  
Powdery Mildew Fungus ◽  
Nonspecific Resistance ◽  
Knock Down ◽  
Highly Efficient

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE proteinin three types of durable, race-nonspecific resistance was observed.

scholarly journals Mutant resources for functional genomics in Dictyostelium discoideum using REMI-seq technology

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Nicole Gruenheit ◽  
Amy Baldwin ◽  
Balint Stewart ◽  
Sarah Jaques ◽  
Thomas Keller ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
High Throughput ◽  
Gene Function ◽  
Evolutionary Biology ◽  
Large Scale ◽  
Function Analysis ◽  
Model Systems ◽  
Genome Wide ◽  
The Social ◽  
Drug Resistance Marker

Abstract Background Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. Results We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of ‘barcoded’ mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. Conclusions We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.

scholarly journals A Reporter System for Reverse Transfection Cell Arrays

2003 ◽  
Vol 8 (6) ◽  
pp. 620-623 ◽  
Author(s):  
Brian L. Webb ◽  
Begoña Díaz ◽  
G. Steven Martin ◽  
Fang Lai
Keyword(s):  
Map Kinase ◽  
High Throughput ◽  
Gene Function ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Signaling Proteins ◽  
Kinase Signaling ◽  
Reporter System ◽  
Map Kinase Signaling ◽  
Reverse Transfection

The incredible speed of gene cloning and sequencing brought about by the genomic revolution has begun to outpace conven tional gene discovery approaches in the pharmaceutical industry. High-throughput approaches for studying gene function in vivo are greatly needed. One potential answer to this challenge is reverse transfection, a high-throughput gene expression method for examining the function of hundreds to thousands of genes in parallel. One limitation of reverse transfection tech nology is the need for posttransfection processing of the arrays to analyze the activity of the expressed proteins. The authors have investigated the use of a reporter construct cotransfected with other genes of interest to monitor and screen gene function on reverse transfection microarrays. They developed a serum response element (SRE) reporter linked to the green fluorescent protein (GFP) that is cotransfected with target genes on reverse transfection arrays for monitoring mitogen-activated protein (MAP) kinase signaling by multiple targets in parallel. The authors show that this reporter system is able to detect inhibition of upstream MAP kinase signaling proteins by the MEK inhibitor U0126. The ability to monitor the activity of multiple signaling proteins in a multiwell format suggests the utility of reverse transfection reporter arrays for high-throughput screening applications.

scholarly journals pOPIN-GG: A resource for modular assembly in protein expression vectors

2021 ◽  
Author(s):  
Adam R Bentham ◽  
Mark Youles ◽  
Melanie N Mendel ◽  
Freya A Varden ◽  
Juan Carlos De la Concepcion ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
Protein Function ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Restriction Enzymes ◽  
Expression Vectors ◽  
Vector Systems ◽  
Protein Expression And Purification ◽  
Modular Cloning

The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials.

Induction of virus-induced gene silencing and in planta validation in cucurbits using the CFMMV-Cm vector

2021 ◽  
Author(s):  
Gung Pyo Lee ◽  
Sun-Ju Rhee ◽  
Yoon Jeong Jang ◽  
Jun-Young Park
Keyword(s):  
Gene Silencing ◽  
Male Sterility ◽  
Differentially Expressed Genes ◽  
High Throughput ◽  
Gene Function ◽  
De Novo ◽  
The Novel ◽  
Virus Induced Gene Silencing ◽  
Male Sterile ◽  
High Throughput Analysis

Virus-induced gene silencing (VIGS) has been employed for the high-throughput analysis of endogenous gene function. We developed a CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) for the efficient generation of viral transcripts in plants. Using the novel pCF93 vector, we identified genes related to male sterility in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. We previously reported reference-based and de novo transcriptomic profiling for the detection of differentially expressed genes between a male fertile line (DAH3615) and its near isogenic male sterile line (DAH3615-MS). Based on the RNA-seq results, we identified 38 de novo-exclusive differentially expressed genes (DEDEGs) that are potentially responsible for male sterility. Partial genes of 200~300bp were cloned into pCF93 which was then inoculated into DAH, a small type of watermelon that enables high-throughput screening with a small cultivation area. In this manner, we simultaneously characterized phenotypes associated with the 38 candidate genes in a common-sized greenhouse. Eight out of the 38 gene-silenced plants produced male sterile flowers with abnormal stamens and no pollens. Gene expression levels in flowers were validated via RT-qPCR. Stamen histological sections from male sterile floral buds and mature flowers showed developmental disruption and shrunken pollen sacs. Based on the current findings, we believe that the novel pCF93 vector and our VIGS system facilitate high-throughput analysis for the study of gene function in watermelons.

scholarly journals Zebrafish Models of Autosomal Dominant Ataxias

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 421
Author(s):  
Ana Quelle-Regaldie ◽  
Daniel Sobrido-Cameán ◽  
Antón Barreiro-Iglesias ◽  
María Jesús Sobrido ◽  
Laura Sánchez
Keyword(s):  
High Throughput ◽  
Gene Function ◽  
Autosomal Dominant ◽  
Neuronal Damage ◽  
Fragile X ◽  
Repeat Expansion ◽  
Cerebellar Dysfunction ◽  
External Fertilization ◽  
Locomotor Deficits ◽  
Neuronal Disorders

Hereditary dominant ataxias are a heterogeneous group of neurodegenerative conditions causing cerebellar dysfunction and characterized by progressive motor incoordination. Despite many efforts put into the study of these diseases, there are no effective treatments yet. Zebrafish models are widely used to characterize neuronal disorders due to its conserved vertebrate genetics that easily support genetic edition and their optic transparency that allows observing the intact CNS and its connections. In addition, its small size and external fertilization help to develop high throughput assays of candidate drugs. Here, we discuss the contributions of zebrafish models to the study of dominant ataxias defining phenotypes, genetic function, behavior and possible treatments. In addition, we review the zebrafish models created for X-linked repeat expansion diseases X-fragile/fragile-X tremor ataxia. Most of the models reviewed here presented neuronal damage and locomotor deficits. However, there is a generalized lack of zebrafish adult heterozygous models and there are no knock-in zebrafish models available for these diseases. The models created for dominant ataxias helped to elucidate gene function and mechanisms that cause neuronal damage. In the future, the application of new genetic edition techniques would help to develop more accurate zebrafish models of dominant ataxias.

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