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Constructing Strong Cell Type-Specific Promoters Through Informed Design

  • Adam J. BrownEmail author
  • David C. James
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1651)

Abstract

Promoter functionality is highly context dependent, as exemplified by gene-specific expression profiles across different tissues and cell types. Cell type-specific promoter regulation is a function of each cell’s unique complement of transcriptional machinery components. Accordingly, to achieve high levels of transcriptional activity within a particular cell type, synthetic promoters must be specifically designed to harness those cells discrete repertoire of available transcription factors. Here, we describe a method for constructing very strong cell type-specific synthetic promoters for use in any given mammalian host cell. Transcription factor regulatory elements (TFREs; or transcription factor binding sites) that can independently mediate activation of recombinant gene transcription in the chosen host cells by using available transcription factor activity are identified and utilized as building blocks to construct novel promoter sequences with varying activities. Bioinformatics analysis of synthetic promoter’s TFRE compositions is then performed to determine how differing relative TFRE abundances explain variations in relative promoter activities. This information is used to derive an optimal second-generation promoter library construction design space, such that promoters with maximal transcriptional activity in the host cell type can be created.

Key words

Synthetic promoters Cell type-specific Mammalian cells Transcriptional regulation Transcription factor binding sites 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Chemical and Biological EngineeringUniversity of SheffieldSheffieldUK

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