PT  - JOURNAL ARTICLE
AU  - Michaels, Yale S.
AU  - Barnkob, Mike B.
AU  - Barbosa, Hector
AU  - Baeumler, Toni A.
AU  - Thompson, Mary K.
AU  - Andre, Violaine
AU  - Colin-York, Huw
AU  - Fritzsche, Marco
AU  - Gileadi, Uzi
AU  - Sheppard, Hilary M.
AU  - Knapp, David J.H.F.
AU  - Milne, Thomas A.
AU  - Cerundolo, Vincenzo
AU  - Fulga, Tudor A.
TI  - Precise tuning of gene expression output levels in mammalian cells
AID  - 10.1101/352377
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 352377
4099  - http://biorxiv.org/content/early/2018/06/20/352377.short
4100  - http://biorxiv.org/content/early/2018/06/20/352377.full
AB  - 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.