A 'poly-transfection' method for rapid, one-pot characterization and optimization of genetic systems

Nucleic Acids Research
Jeremy J GamRon Weiss

Abstract

Biological research is relying on increasingly complex genetic systems and circuits to perform sophisticated operations in living cells. Performing these operations often requires simultaneous delivery of many genes, and optimizing the stoichiometry of these genes can yield drastic improvements in performance. However, sufficiently sampling the large design space of gene expression stoichiometries in mammalian cells using current methods is cumbersome, complex, or expensive. We present a 'poly-transfection' method as a simple yet high-throughput alternative that enables comprehensive evaluation of genetic systems in a single, readily-prepared transfection sample. Each cell in a poly-transfection represents an independent measurement at a distinct gene expression stoichiometry, fully leveraging the single-cell nature of transfection experiments. We first benchmark poly-transfection against co-transfection, showing that titration curves for commonly-used regulators agree between the two methods. We then use poly-transfections to efficiently generate new insights, for example in CRISPRa and synthetic miRNA systems. Finally, we use poly-transfection to rapidly engineer a difficult-to-optimize miRNA-based cell classifier for discrim...Continue Reading

References

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Citations

Nov 5, 2020·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Hanieh MoradianManfred Gossen
Feb 19, 2021·Expert Opinion on Drug Delivery·Dmitrii S LinnikAlexander S Timin
May 13, 2021·PeerJ·Zhi Xiong ChongWan Yong Ho
Jun 18, 2021·Cell Systems·Nika ShakibaDomitilla Del Vecchio

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Methods Mentioned

BETA
transfection
transfections
FlowSeq
flow cytometry

Software Mentioned

CyTOF
MATLAB
Addgene
FlowSeq

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