Brown Adam J, James David C
Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, England, UK.
Methods Mol Biol. 2017;1651:131-145. doi: 10.1007/978-1-4939-7223-4_10.
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.
启动子功能高度依赖于上下文，不同组织和细胞类型中的基因特异性表达谱就是例证。细胞类型特异性启动子调控是每个细胞独特的转录机制成分组合的一种功能。因此，为了在特定细胞类型中实现高水平的转录活性，必须专门设计合成启动子，以利用这些细胞中可用转录因子的离散库。在此，我们描述了一种构建非常强大的细胞类型特异性合成启动子的方法，用于任何给定的哺乳动物宿主细胞。通过利用可用转录因子活性，鉴定出能够在所选宿主细胞中独立介导重组基因转录激活的转录因子调控元件（TFREs；或转录因子结合位点），并将其用作构建具有不同活性的新型启动子序列的构建模块。然后对合成启动子的TFRE组成进行生物信息学分析，以确定不同的相对TFRE丰度如何解释相对启动子活性的变化。这些信息用于推导最佳的第二代启动子文库构建设计空间，从而能够创建在宿主细胞类型中具有最大转录活性的启动子。
