Engineering Molecular Sensors for Stem Cell Function

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信息的高灵敏度探针。特别是，基于荧光和发光蛋白质的成像遗传编码传感器，可以永久引入细胞和生物体，可以产生关于许多细胞过程的详细信息，如信号转导和基因调控。这种传感器具有巨大的潜力，可以提供数据，帮助研究复杂的生物机制和疾病病理。然而，在实现其全部潜力之前，必须改进传感器性能，并且必须构建模型系统来测试和突出其实用性。 分子传感器将被开发用于研究具有医学潜力的重要模型系统，成体神经干细胞。这个系统将被用来强调的想法，复杂的基因调控和信号转导机制，翻译细胞外信号到细胞内的决定，可以阐明只有通过在这些过程中，在真实的时间和在单细胞水平的多个路口采取测量。萤火虫荧光素酶报告将被开发，以量化参与两个神经干细胞命运决定，自我更新和星形胶质细胞分化，由Shh和Notch信号控制的关键转录因子的调节。同时，荧光蛋白传感器将报告干细胞的命运承诺。将使用免疫荧光和定量PCR方法验证传感器功能。最后，为了进一步改善萤火虫荧光素酶报告基因的特性，将采用基于DNA改组的高通量定向进化方法来创建具有更高灵敏度的新型荧光素酶变体。 我们的具体目标是： 1)利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改变干细胞的命运决定 2)为了确定是否可以使用定向进化方法产生更高灵敏度的荧光素酶变体。

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.