Engineering Molecular Sensors for Stem Cell Function

用于干细胞功能的工程分子传感器

• 批准号: 6879693
• 负责人: DAVID V SCHAFFER
• 金额: $ 16.8万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879693
• 关键词: astrocytes; bioengineering /biomedical engineering; biological signal transduction; biosensor device; cell differentiation; directed evolution; genetic regulation; high throughput technology; immunofluorescence technique; luciferin monooxygenase; molecular probes; polymerase chain reaction; stem cells; time resolved data; transcription factor

DESCRIPTION (provided by applicant): Molecular sensors are high sensitivity probes capable of reporting quantitative biological information at the molecular and cellular levels. In particular, imaging genetically encoded sensors based on fluorescent and luminescent proteins, which can be permanently introduced into cells and organisms, can yield detailed information on numerous cellular processes such as signal transduction and gene regulation. Such sensors have enormous potential for providing data to aid in the investigation of complex biological mechanisms and disease pathologies. Before their full potential can be realized, however, sensor properties must be improved, and model systems must be constructed to test and highlight their utility. Molecular sensors will be developed to study an important model system with medical potential, adult neural stem cells. This system will be employed to emphasize the idea that the complex gene regulation and signal transduction mechanisms that translate extracellular signals into intracellular decisions can be elucidated only by taking measurements at multiple junctures in these processes, in real time and at the single cell level. Firefly luciferase reporters will be developed to quantify the regulation of key transcription factors involved in two neural stem cell fate decisions, self-renewal and astrocytic differentiation, controlled by Shh and Notch signaling. Simultaneously, fluorescent protein sensors will report stem cell fate commitment. Sensor function will be validated using immunofluorescence and quantitative PCR methods. Finally, to further improve the properties of firefly luciferase reporters, a high throughput directed evolution approach based on DNA shuffling will be employed to create novel luciferase variants with higher sensitivity. Our Specific Aims are: 1) To employ molecular sensors to determine at the single cell level whether threshold levels of extracellular signals switch stem cell fate decisions 2) To determine whether higher sensitivity variants of luciferase can be created using a directed evolution approach.

描述（由申请人提供）：分子传感器是能够在分子和细胞水平上报告定量生物学信息的高灵敏度探针。特别是，基于荧光和发光蛋白的遗传编码传感器，可以永久引入细胞和生物，可以在众多细胞过程中产生详细信息，例如信号转导和基因调节。此类传感器具有提供数据以帮助研究复杂生物学机制和疾病病理学的巨大潜力。但是，在实现其全部潜力之前，必须提高传感器属性，并且必须构建模型系统以测试和突出其实用性。 将开发分子传感器来研究具有成年神经干细胞的重要模型系统。该系统将被用来强调这样的想法：将细胞外信号转化为细胞内决策的复杂基因调节和信号转导机制只能通过在这些过程中，实时和单个细胞水平上进行多个连接处进行测量来阐明细胞内决策。将开发萤火虫荧光素酶记者，以量化由SHH和Notch信号传导控制的两个神经干细胞命运决策的关键转录因子的调节，即自我更新和星形细胞分化。同时，荧光蛋白传感器将报告干细胞命运的承诺。传感器功能将使用免疫荧光和定量PCR方法验证。最后，为了进一步提高萤火虫荧光素酶报告器的特性，将采用基于DNA洗牌的高吞吐量定向进化方法来创建具有较高灵敏度的新型荧光素酶变体。 我们的具体目的是： 1）使用分子传感器在单细胞水平上确定细胞外信号的阈值水平是否开关干细胞命运决定 2）确定是否可以使用定向进化方法来创建荧光素酶的更高灵敏度变体。