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 Sorting out antibiotics' mechanisms of action: a double fluorescent protein reporter for high throughput screening of ribosome and DNA biosynthesis inhibitors

2016 ◽  
pp. AAC.02117-16 ◽  
Author(s):  
Ilya A. Osterman ◽  
Ekaterina S. Komarova ◽  
Dmitry I. Shiryaev ◽  
Ilya A. Korniltsev ◽  
Irina M. Khven ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cost Effective ◽  
High Signal ◽  
Red Fluorescent Protein ◽  
Reporter System ◽  
Ribosome Stalling

In order to accelerate drug discovery, a simple, reliable and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double reporter system for not only antimicrobial activity detection, but also for simultaneous sorting of potential antimicrobials into those that cause ribosome stalling, and others that induce SOS response due to DNA damage. In this reporter system the red fluorescent protein generfpwas placed under the control of the SOS-induciblesulApromoter. The far-red fluorescent protein genekatushka2Swas inserted downstream the tryptophan attenuator where two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator, to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to any ribosome stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need in enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.

scholarly journals Reassembling green fluorescent protein for in vitro evaluation of trans-translation

2020 ◽  
Vol 48 (4) ◽  
pp. e22-e22
Author(s):  
Charlotte Guyomar ◽  
Marion Thépaut ◽  
Sylvie Nonin-Lecomte ◽  
Agnès Méreau ◽  
Renan Goude ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Messenger Rna ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Chemical Compounds ◽  
Reporter System ◽  
New Antibiotics ◽  
Green Fluorescent

Abstract In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.

The p21-activated kinase PAK 5 is involved in formalin-induced nociception through regulation of MAP-kinase signaling and formalin-specific receptors

2012 ◽  
Vol 234 (1) ◽  
pp. 121-128 ◽  
Author(s):  
Ovidiu Coste ◽  
Christine V. Möser ◽  
Marco Sisignano ◽  
Katharina L. Kynast ◽  
Audrey Minden ◽  
...  
Keyword(s):  
Map Kinase ◽  
Kinase Signaling ◽  
Map Kinase Signaling ◽  
Specific Receptors ◽  
P21 Activated Kinase

scholarly journals Specificity of MAP Kinase Signaling in Yeast Differentiation Involves Transient versus Sustained MAPK Activation

2001 ◽  
Vol 8 (3) ◽  
pp. 683-691 ◽  
Author(s):  
Walid Sabbagh ◽  
Laura J Flatauer ◽  
A.Jane Bardwell ◽  
Lee Bardwell
Keyword(s):  
Map Kinase ◽  
Kinase Signaling ◽  
Mapk Activation ◽  
Map Kinase Signaling

scholarly journals Non-invasive and high-throughput interrogation of exon-specific isoform expression

2021 ◽  
Author(s):  
Dong-Jiunn Jeffery Truong ◽  
Teeradon Phlairaharn ◽  
Bianca Eßwein ◽  
Christoph Gruber ◽  
Deniz Tümen ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
High Sensitivity ◽  
Therapeutic Interventions ◽  
Dominant Factor ◽  
Reporter System ◽  
Isoform Expression ◽  
Induced Pluripotent

AbstractExpression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

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