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A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.

ABSTRACT: Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50,000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.

SUBMITTER: Choe J

PROVIDER: S-EPMC1065264 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.

Choe Juno J Guo Haiwei H HH van den Engh Ger G

Nucleic acids research 20050314 5

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50,000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controllin ...[more]

PMID: 15767274

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Project description:In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of the highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CLpro 3CLpro is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this work, we report a new assay to identify inhibitors of 3CLpro Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CLpro This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread.IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CLpro This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.

Dataset Information

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.

Publications

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.

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