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Precise tuning of gene expression output levels in mammalian cells

View ORCID ProfileYale S. Michaels, Mike B Barnkob, Hector Barbosa, Toni A Baeumler, View ORCID ProfileMary K Thompson, Violaine Andre, Huw Colin-York, Marco Fritzsche, Uzi Gileadi, Hilary M Sheppard, David JHF Knapp, Thomas A Milne, Vincenzo Cerundolo, Tudor A Fulga
doi: https://doi.org/10.1101/352377
Yale S. Michaels
Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford;
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Mike B Barnkob
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS;
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Hector Barbosa
Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford;
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Toni A Baeumler
Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford;
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Mary K Thompson
Department of Biochemistry, University of Oxford, OX1 3QU, Oxford United Kingdom;
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Violaine Andre
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS;
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Huw Colin-York
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS;
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Marco Fritzsche
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS;
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Uzi Gileadi
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS;
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Hilary M Sheppard
School of Biological Sciences, University of Auckland, Auckland, 1050, New Zealand;
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David JHF Knapp
Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford;
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Thomas A Milne
WIMM, MRC Molecular Haematology Unit, NIHR Oxford Biomedical Research Centre Programme
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Vincenzo Cerundolo
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS;
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Tudor A Fulga
Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford;
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  • For correspondence: tudor.fulga@imm.ox.ac.uk
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Abstract

Precise, analogue regulation of gene expression is critical for development, homeostasis and regeneration in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity, while RNA interference (RNAi) can lead to pervasive off-target effects and unpredictable levels of repression. Here we report on a method for the precise control of gene expression levels in mammalian cells based on engineered, synthetic microRNA response elements (MREs). To develop this system, we established a high-throughput sequencing approach for measuring the efficacy of thousands of miR-17 MRE variants. This allowed us to create a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to control the expression of user specified genes. To demonstrate the value of this technology, we used a panel of miSFITs to tune the expression of a peptide antigen in a mouse melanoma model. This analysis revealed that antigen expression level is a key determinant of the anti-tumour immune response in vitro and in vivo. miSFITs are a powerful tool for modulating gene expression output levels with applications in research and cellular engineering.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 20, 2018.
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Precise tuning of gene expression output levels in mammalian cells
Yale S. Michaels, Mike B Barnkob, Hector Barbosa, Toni A Baeumler, Mary K Thompson, Violaine Andre, Huw Colin-York, Marco Fritzsche, Uzi Gileadi, Hilary M Sheppard, David JHF Knapp, Thomas A Milne, Vincenzo Cerundolo, Tudor A Fulga
bioRxiv 352377; doi: https://doi.org/10.1101/352377
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Precise tuning of gene expression output levels in mammalian cells
Yale S. Michaels, Mike B Barnkob, Hector Barbosa, Toni A Baeumler, Mary K Thompson, Violaine Andre, Huw Colin-York, Marco Fritzsche, Uzi Gileadi, Hilary M Sheppard, David JHF Knapp, Thomas A Milne, Vincenzo Cerundolo, Tudor A Fulga
bioRxiv 352377; doi: https://doi.org/10.1101/352377

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