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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 J.H.F. Knapp, Thomas A. Milne, Vincenzo Cerundolo, Tudor A. Fulga
doi: https://doi.org/10.1101/352377
Yale S. Michaels
1Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom
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  • ORCID record for Yale S. Michaels
Mike B. Barnkob
2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS Oxford, United Kingdom
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Hector Barbosa
1Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom
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Toni A. Baeumler
1Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom
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Mary K. Thompson
3Department of Biochemistry, University of Oxford, OX1 3QU, Oxford United Kingdom
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Violaine Andre
2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS Oxford, United Kingdom
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Huw Colin-York
2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS Oxford, United Kingdom
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Marco Fritzsche
2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS Oxford, United Kingdom
4Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, OX3 7FY, Oxford United Kingdom
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Uzi Gileadi
2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS Oxford, United Kingdom
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Hilary M. Sheppard
5School of Biological Sciences, University of Auckland, Auckland, 1050, New Zealand
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David J.H.F. Knapp
1Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom
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Thomas A. Milne
6Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford OX3 9DS, UK
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Vincenzo Cerundolo
2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, OX3 9DS Oxford, United Kingdom
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Tudor A. Fulga
1Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom
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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 J.H.F. 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 J.H.F. Knapp, Thomas A. Milne, Vincenzo Cerundolo, Tudor A. Fulga
bioRxiv 352377; doi: https://doi.org/10.1101/352377

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