Viral Protease Assay Based on GAL4 Inactivation Is Applicable to High-Throughput Screening in Mammalian Cells

1999 ◽  
Vol 269 (1) ◽  
pp. 133-138 ◽  
Author(s):  
Joseph F. Lawler ◽  
Solomon H. Snyder
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Viral Protease ◽  
Protease Assay

scholarly journals Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

2009 ◽  
Vol 53 (11) ◽  
pp. 4825-4834 ◽  
Author(s):  
Kao-Lu Pan ◽  
Jin-Ching Lee ◽  
Hsing-Wen Sung ◽  
Teng-Yuang Chang ◽  
John T.-A. Hsu
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reporter System ◽  
Viral Protease ◽  
Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

High throughput screening methods for freeze-drying of fungi and mammalian cells

Cryobiology ◽  
2016 ◽  
Vol 73 (3) ◽  
pp. 425
Author(s):  
P. Kilbride ◽  
E. Thompson ◽  
M. Ryan ◽  
K. Mahbubani ◽  
B. Luke ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Freeze Drying ◽  
Screening Methods

High-throughput screening and selection of mammalian cells for enhanced protein production

2016 ◽  
Vol 11 (7) ◽  
pp. 853-865 ◽  
Author(s):  
Joseph J. Priola ◽  
Nathan Calzadilla ◽  
Martina Baumann ◽  
Nicole Borth ◽  
Christopher G. Tate ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Protein Production ◽  
Selection Of

scholarly journals A Thallium-Sensitive, Fluorescence-Based Assay for Detecting and Characterizing Potassium Channel Modulators in Mammalian Cells

2004 ◽  
Vol 9 (8) ◽  
pp. 671-677 ◽  
Author(s):  
C. David Weaver ◽  
David Harden ◽  
Steven I. Dworetzky ◽  
Barbara Robertson ◽  
Ronald J. Knox
Keyword(s):  
Potassium Channels ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Ease Of Use ◽  
Functional Assay ◽  
Chemical Libraries ◽  
Potassium Channel Modulators ◽  
Small Molecule Modulators

Potassium channels have been identified as targets for a large number of therapeutic indications. The ability to use a high-throughput functional assay for the detection and characterization of small-molecule modulators of potassium channels is very desirable. However, present techniques capable of screening very large chemical libraries are limited in terms of data quality, temporal resolution, ease of use, and requirements for specialized instrumentation. To address these issues, the authors have developed a fluorescence-based thalliumflux assay. This assay is capable of detectingmodulators of both voltageand ligand-gated potassium channels expressed inmammalian cells. The thalliumflux assay can use instruments standard to most high-throughput screening laboratories, and using such equipment has been successfully employed to screen large chemical libraries consisting of hundreds of thousands of compounds.

High Throughput Screening of Gene Functions in Mammalian Cells Using Reversely Transfected Cell Arrays: Review And Protocol

2008 ◽  
Vol 11 (2) ◽  
pp. 159-172 ◽  
Author(s):  
Michael Sturzl ◽  
Andreas Konrad ◽  
Gaby Sander ◽  
Effi Wies ◽  
Frank Neipel ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Gene Functions ◽  
Cell Arrays

scholarly journals Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast

2016 ◽  
Vol 21 (4) ◽  
pp. 381-390 ◽  
Author(s):  
Yujian Zhang ◽  
Defeng Tian ◽  
Hironori Matsuyama ◽  
Takashi Hamazaki ◽  
Takayuki Shiratsuchi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Drug Targets ◽  
Energy Homeostasis ◽  
Adenine Nucleotide ◽  
Screening Strategy ◽  
Adenine Nucleotide Translocase ◽  
Specific Expression ◽  
Knock Out

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast “knock-out/knock-in” screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.

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