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Probing the characteristics of genetic circuits integrated in mammalian cells and

探究哺乳动物细胞中整合的遗传电路的特征

基本信息

批准号：
8180749
负责人：
Leonidas Bleris
金额：
$ 30.6万
依托单位：
UNIVERSITY OF TEXAS DALLAS
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8180749
关键词：
Address Architecture Behavior Beryllium Biological Biological Models Biology Cell physiology Cells Characteristics Complex Computer Architectures DNA Data Data Analyses Dependence Detection Diagnosis Diagnostic Disease Dosage Compensation (Genetics)Elements Engineering Environment Family Feedback Flow Cytometry Gene Dosage Generations Genes Genetic Genetic Template Genetic Transcription Goals Health Healthcare Hereditary Disease Human Biology In Situ Individual Institution Investigation Lead Libraries Life Light Logic Malignant Neoplasms Mammalian Cell Mathematics Measurement Medical Medicine Methods MicroRNAs Microscopy Molecular Monitor Names Noise Open Reading Frames Organ Organism Output Pathway interactions Population Prevention Process Property Proteins Protocols documentation Public Health Research Scientist Signal Transduction Solutions Students Synthetic Genes System Techniques Testing Therapeutic Time Tissues Variant Viral Work base biological systems experience feeding graduate student human disease information processing insight novel operation prevent programs promoter prototype recombinase research study sensor stoichiometry synthetic biology theories tool

项目摘要

DESCRIPTION (provided by applicant): Complex combinations of molecular signals in cells are an excellent indicator of multi-gene disorders, including cancers and hereditary diseases. A system capable to detect these conditions may be used as a highly selective tool for diagnosis, prevention, treatment, and monitoring at a single-cell level in ways that achieve optimal and highly specific health-care. Towards this direction, scientists have developed first generation genetic circuits that operate as information-processing systems. However, the utility and scalability of these prototypes is hampered by fluctuations in stoichiometry between different components of the circuit in individual cells. Therefore, it is critical to construct sophisticated expression units whose gene product will depend only weakly on the number of unit copies in a cell and on the global transcription efficiency. Such stand-alone units could then be combined into networks that could be expected to function reliably in the face of large internal fluctuations. We argue that particular network architectures (or topologies) may provide the solution towards this goal. The number of possible topologies for a given set of pathway elements is large and it grows exponentially with the number of elements, making their exhaustive investigation intangible. Fortunately, recent research has uncovered that certain topologies appear more frequently than others. Those topologies, named "network motifs", are composed of relatively few elements and are embedded as "modules" or "nodes" in larger networks and pathways. Based on preliminary experiments, we form the hypothesis that specific families of biological network motifs can be used to reduce noise and fluctuations in intracellular activity and most importantly, the copy number variability. Further investigation of these results with the proposed experiments can radically change the field and lead to several health-related diagnostic and therapeutic applications. More generally, as many human diseases are essentially network-level phenomena, unraveling properties of biological motifs is central to understanding human biology. Our long-term objective is to construct functional and scalable synthetic gene circuits able to perform predetermined functions in the face of large internal fluctuations. The proposed aims will bring us considerably closer to this objective. More specifically, we aim to construct and integrate in mammalian cells a range of feedback and feedforward motif circuits, utilizing a library of building blocks and using both viral delivery and recombinase systems. In order to test our hypothesis, we will characterize the noise and copy number dependence of the genetic circuits using microscopy and flow cytometry measurements. Finally, we propose to implement a first generation of genetic circuits for detection and monitoring of endogenous miRNA signals. We aim to show that the use of the aforementioned topologies in the circuits renders them suitable for high- throughput monitoring and yields increased accuracy in the miRNA sensing. PUBLIC HEALTH RELEVANCE: We propose a comprehensive characterization of specific circuit architectures and their implementation in first generation sensors for endogenous signal detection and monitoring. The results will spark a wide range of applications relevant to public health and specific to monitoring and processing intracellular signals in a reliable manner.

描述（由申请人提供）：细胞中分子信号的复杂组合是多基因疾病的良好指标，包括癌症和遗传性疾病。能够检测这些疾病的系统可作为一种高度选择性的工具，用于单细胞水平的诊断、预防、治疗和监测，从而实现最佳和高度特异性的卫生保健。朝着这个方向，科学家们已经开发出了作为信息处理系统运行的第一代遗传电路。然而，这些原型的实用性和可扩展性受到单个细胞中电路不同组件之间化学计量波动的阻碍。因此，构建复杂的表达单元至关重要，其基因产物仅弱依赖于细胞中的单位拷贝数和全局转录效率。然后，这些独立的单位可以组合成网络，在面对巨大的内部波动时，可以预期这些网络能够可靠地运行。我们认为，特定的网络体系结构（或拓扑结构）可能为实现这一目标提供解决方案。对于给定的一组路径元素，可能的拓扑数量很大，并且随着元素的数量呈指数增长，使得它们的详尽研究变得不可捉摸。幸运的是，最近的研究发现，某些拓扑比其他拓扑出现得更频繁。这些拓扑结构被称为“网络基序”，由相对较少的元素组成，并作为“模块”或“节点”嵌入到更大的网络和路径中。基于初步实验，我们形成了一个假设，即特定的生物网络基序家族可以用来减少细胞内活动的噪声和波动，最重要的是，拷贝数可变性。对这些实验结果的进一步研究可以从根本上改变这个领域，并导致一些与健康相关的诊断和治疗应用。更一般地说，由于许多人类疾病本质上是网络层面的现象，揭示生物基序的特性是理解人类生物学的核心。我们的长期目标是构建功能性和可扩展的合成基因电路，能够在面对巨大的内部波动时执行预定功能。拟议的目标将使我们大大接近这一目标。更具体地说，我们的目标是在哺乳动物细胞中构建和整合一系列反馈和前馈基序电路，利用构建模块库并使用病毒传递和重组酶系统。为了验证我们的假设，我们将使用显微镜和流式细胞术测量来表征遗传电路的噪声和拷贝数依赖性。最后，我们建议实现用于检测和监测内源性miRNA信号的第一代遗传电路。我们的目标是证明在电路中使用上述拓扑结构使它们适合于高通量监测，并提高miRNA传感的准确性。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Assembly and validation of versatile transcription activator-like effector libraries.

DOI：
10.1038/srep04857
发表时间：
2014-05-06
期刊：
Scientific reports
影响因子：
4.6
作者：
Li Y;Ehrhardt K;Zhang MQ;Bleris L
通讯作者：
Bleris L

Reverse engineering validation using a benchmark synthetic gene circuit in human cells.

DOI：
10.1021/sb300093y
发表时间：
2013-05-17
期刊：
ACS SYNTHETIC BIOLOGY
影响因子：
4.7
作者：
Kang, Taek;White, Jacob T.;Xie, Zhen;Benenson, Yaakov;Sontag, Eduardo;Bleris, Leonidas
通讯作者：
Bleris, Leonidas

Transcripts for combined synthetic microRNA and gene delivery.