Precision controllers of mammalian gene expression

项目摘要/摘要 我们将建立这样的系统，允许大大改进对基因表达的化学调节控制 哺乳动物细胞，并将其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，这通常是通过一种特殊的方法来完成的，即由四环素类似物诱导 Tn10 TetR衍生物驱动的基因表达。TetR衍生品及其使用原则 从20世纪90年代初开始。这些有20多年历史的方法继续帮助癌蛋白、肿瘤的研究 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。 但这些方法受到了严重的限制。首先，常用的系统在运营中产生 要么全有要么全无的归纳，而不是可调的表达。其次，TetR是唯一一个开发得很好的 化学诱导的DNA结合部分被广泛使用，因此，研究人员通常可以 只能有条件地诱导一个基因产物或触发一个事件。最后，这些系统不使用封闭式 循环或反馈控制。因此，诱导的基因表达受到显著的细胞间差异的影响， 由于细胞内基因表达能力的差异，G. 现在，许多发展使我们能够建立不受这些限制的系统。考虑 细菌系统中的反馈控制，更好地理解原核生物阻遏因子的人工抑制 在真核细胞中(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 可调诱导对细胞间变异有缓冲作用。大规模细菌DNA测序揭示了 数千个新的TetR家族抑制物来制造新的蛋白质。CAS9/CRISPR方法现在促进了 通过比较来自不同测试的表达式来优化性能的实验 整合在同一哺乳动物基因组位置的单一拷贝的结构。 在这个试点阶段的工作中，我们将集中利用酵母经验教训来构建更好的风险 哺乳动物基因表达的控制者。我们将至少开发并分发给其他研究人员 三(3)个新的化学可调的哺乳动物控制器，对不同的小分子配体做出反应。 我们预计这些控制器将在癌症研究中广泛使用，使(在许多情况下) 例)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)更严格地界定引起生物效应的蛋白质的阈值水平；c)优化时机和 多种转录因子的表达水平，以最好地产生特定的分化细胞类型d) 用于抗癌药物筛选的癌蛋白表达受控细胞系的构建。

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.