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Quantitative and multiplexed chemical-genetic phenotyping in mammalian cells with QMAP-Seq.

ABSTRACT: Chemical-genetic interaction profiling in model organisms has proven powerful in providing insights into compound mechanism of action and gene function. However, identifying chemical-genetic interactions in mammalian systems has been limited to low-throughput or computational methods. Here, we develop Quantitative and Multiplexed Analysis of Phenotype by Sequencing (QMAP-Seq), which leverages next-generation sequencing for pooled high-throughput chemical-genetic profiling. We apply QMAP-Seq to investigate how cellular stress response factors affect therapeutic response in cancer. Using minimal automation, we treat pools of 60 cell types-comprising 12 genetic perturbations in five cell lines-with 1440 compound-dose combinations, generating 86,400 chemical-genetic measurements. QMAP-Seq produces precise and accurate quantitative measures of acute drug response comparable to gold standard assays, but with increased throughput at lower cost. Moreover, QMAP-Seq reveals clinically actionable drug vulnerabilities and functional relationships involving these stress response factors, many of which are activated in cancer. Thus, QMAP-Seq provides a broadly accessible and scalable strategy for chemical-genetic profiling in mammalian cells.

SUBMITTER: Brockway S

PROVIDER: S-EPMC7661543 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Quantitative and multiplexed chemical-genetic phenotyping in mammalian cells with QMAP-Seq.

Brockway Sonia S Wang Geng G Jackson Jasen M JM Amici David R DR Takagishi Seesha R SR Clutter Matthew R MR Bartom Elizabeth T ET Mendillo Marc L ML

Nature communications 20201112 1

Chemical-genetic interaction profiling in model organisms has proven powerful in providing insights into compound mechanism of action and gene function. However, identifying chemical-genetic interactions in mammalian systems has been limited to low-throughput or computational methods. Here, we develop Quantitative and Multiplexed Analysis of Phenotype by Sequencing (QMAP-Seq), which leverages next-generation sequencing for pooled high-throughput chemical-genetic profiling. We apply QMAP-Seq to i ...[more]

PMID: 33184288

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Project description:BackgroundA variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.ResultsA nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale.ConclusionThe combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.

Dataset Information

Quantitative and multiplexed chemical-genetic phenotyping in mammalian cells with QMAP-Seq.

Publications

Quantitative and multiplexed chemical-genetic phenotyping in mammalian cells with QMAP-Seq.

Similar Datasets

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