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Precision controllers of mammalian gene expression

哺乳动物基因表达的精确控制器

基本信息

批准号：
10602901
负责人：
Roger Brent
金额：
$ 0.23万
依托单位：
FRED HUTCHINSON CANCER CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10602901
关键词：
Precision controllers mammalian gene expression

项目摘要

Project Summary/ Abstract We will build systems that allow greatly improved chemically-tuned control of gene expression in mammalian cells, and make them available to other researchers. Induction of gene expression on demand is a mainstay of modern biology and cancer research. Nowadays, this is commonly accomplished by one particular approach, induction by tetracycline analogs of gene expression driven by Tn10 TetR derivatives. The TetR derivatives and doctrine for using them date from the early 1990s. These 20+ year-old methods continue to aid research on oncoproteins, tumor suppressors, cell death proteins, cell cycle proteins, and other key proteins and RNAs in cells and animals. But these approaches suffer from serious limitations. First, systems in common use operationally produce all-or-nothing induction, rather than tunable expression. Second, TetR is the only well developed chemically-inducible DNA binding moiety in wide use, and, as a consequence, investigators can typically only conditionally induce one gene product or trigger one event. Finally, these systems do not use closed- loop or feedback control. Induced gene expression is thus subject to significant cell-to-cell variation, much of it due to intracellular differences in gene expression capacity, G. A number of developments now allow us to build systems not subject to these limitations. Consideration of feedback control in bacterial systems, better understanding of artificial repression by prokaryotic repressors in eukaryotes (which we developed in the 1980s), and work in yeast has enabled systems that offer tunable induction buffered against cell-cell variation. Wholesale bacterial DNA sequencing has revealed 1000s of new TetR family repressors to make new proteins. Cas9/ CRISPR approaches now facilitate experimentation to optimize performance, by making it possible to compare expression from different test constructs integrated in single copy at the same mammalian genomic site. In this pilot phase of work, we will concentrate the risks of using lessons from yeast to build better controllers of mammalian gene expression. We will develop and distribute to other researchers at least three (3) new chemically tunable mammalian controllers that respond to different small-molecule ligands. We expect that these controllers will become broadly used in cancer research, enabling (among many examples) a) finer control of timing and amount of oncoproteins and other key proteins in cells and animals b) tighter definition of threshold levels of proteins that cause biological effects c) optimization of timing and level of expression of multiple transcription factors to best generate specific differentiated cell types d) construction of cell lines with controlled oncoprotein expression to be used in screens for anticancer drugs.

项目总结/摘要我们将建立一个系统，允许大大改善化学调谐控制基因表达，哺乳动物细胞，并将它们提供给其他研究人员。按需诱导基因表达是现代生物学和癌症研究的支柱。如今，这通常通过一种特定的方法来实现，即通过四环素类似物诱导，由Tn 10 TetR衍生物驱动的基因表达。TetR衍生物和使用它们的原则从90年代初开始这些已有20多年历史的方法继续帮助研究癌蛋白，肿瘤抑制子、细胞死亡蛋白、细胞周期蛋白以及细胞和动物中的其他关键蛋白和RNA。但这些方法有严重的局限性。首先，共同使用的系统在操作上产生全或无归纳，而不是可调表达式。其次，TetR是唯一一个发展良好的化学诱导的DNA结合部分，因此，研究人员通常可以仅有条件地诱导一种基因产物或触发一种事件。最后，这些系统不使用封闭的- 回路或反馈控制。因此，诱导的基因表达受到显著的细胞间变异的影响，由于细胞内基因表达能力的差异，G. 现在的一些发展使我们能够建立不受这些限制的系统。审议反馈控制在细菌系统中，更好地理解人工阻遏原核阻遏在真核生物中（我们在20世纪80年代开发的），在酵母中的工作使系统能够提供可调诱导缓冲细胞间变异。大规模细菌DNA测序显示 1000个新的TetR家族阻遏物来制造新的蛋白质。Cas9/ CRISPR方法现在可以促进实验，以优化性能，使之有可能比较表达从不同的测试在同一哺乳动物基因组位点以单拷贝整合的构建体。在这个试验阶段的工作，我们将集中风险，利用酵母的教训，以建立更好的哺乳动物基因表达的控制者。我们将开发并分发给其他研究人员，至少三（3）新的化学可调哺乳动物控制器，响应不同的小分子配体。我们预计这些控制器将广泛用于癌症研究，使（在许多例子）a）更好地控制细胞和动物中癌蛋白和其他关键蛋白的时间和数量 B）引起生物效应的蛋白质的阈值水平的更严格定义多种转录因子的表达水平以最佳地产生特异性分化细胞类型d）构建具有受控的癌蛋白表达的细胞系以用于筛选抗癌药物。