| Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures
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Author:
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2021-04-20
[Abstract] Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer’s Disease (AD). We have recently developed a novel optically-responsive tool (the ‘CofActor’ system) that couples cofilin and actin (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a ...
[摘要] [摘要]参与神经变性蛋白质可具有耦合光遗传学试剂来创建快速且灵敏的记者到provid Ë洞察介导的神经变性疾病，包括进展的生化过程阿尔茨海默氏病（AD）。我们最近开发了一种新型光学-响应工具（“辅”系统）夫妇COF伊林和行动中使用（与神经退行性疾病相关的早期阶段，细胞骨架异常关键球员）光门控光遗传学 蛋白质提供时空分辨率的氧化和高能应激依赖的生化事件。与目前可用的基于小分子的生物传感器来监测细胞氧化还原环境的变化相比，CofActor是一种光激活的，遗传编码的氧化还原传感器，可以通过精确的空间和时间控制来激活。在这里，我们描述了从新生小鼠制备的解离海马神经元培养物中CofActor系统的表达和激活的协议。将培养物转染用大号ipofectamine上的第五天体外（DIV5），然后暴露于细胞应激诱导刺激，导致的肌动蛋白的形成丝切蛋白可使用活细胞成像技术可以观察到杆。本文所述的方案可用于研究暴露于神经退行性刺激（例如毒性Aβ42低聚物）的活神经元中与压力相关的细胞骨架失调。此外，从AD的转基因小鼠模型和/或与KO相关的小鼠KO小鼠分离的神经元中传感器的表达可以促进我们对与神经变性相关的早期细胞骨架功能障碍的分子基础的理解。
[背景]神经变性疾病的生化标志（神经原纤维，团块和缠结，提高活性氧物质（ROS） ...
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| Novel Method for Site-specific Induction of Oxidative DNA Damage to Study Recruitment of Repair Proteins to Heterochromatin and Euchromatin
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Author:
Date:
2014-06-05
[Abstract] ROS-induced DNA damage is repaired in living cells within a temporal and spatial context, and chromatin structure is critical to a consideration of DNA repair processes in situ. It’s well known that chromatin remodeling factors participate in many DNA damage repair pathways, indicating the importance of chromatin remodeling in facilitating DNA damage repair. To date, there has been no method to induce site-specific oxidative DNA damage in living cells. Therefore, it is not known whether the DNA repair mechanisms differ within active or condensed chromatin. We recently established a novel method, DTG (Damage Targeted at one Genome-site), to study DNA damage response of reactive oxygen species (ROS)-induced DNA damage in living cell at one genome loci with active or inactive ...
[摘要] ROS诱导的DNA损伤在时间和空间背景下在活细胞中修复，并且染色质结构对于原位DNA修复过程的考虑是关键的。众所周知，染色质重塑因子参与许多DNA损伤修复途径，表明染色质重塑促进DNA损伤修复的重要性。到目前为止，还没有方法诱导活细胞中的位点特异性氧化性DNA损伤。因此，不知道DNA修复机制在活性或凝集的染色质中是否不同。我们最近建立了一种新的方法，DTG（损害靶向一个基因组位点），研究活性氧（ROS）诱导的DNA损伤活动细胞中的DNA损伤反应在一个基因组活性或无活性转录。为此，我们在U2OS细胞中在X染色体上整合了四环素响应元件（TRE）盒（〜90kb）（Lan等人，2010），然后融合KillerRed（KR）刺激的ROS诱导物，其可以特异性产生ROS诱导的DNA损伤，tet-阻遏物（tetR-KR，OFF）或转录激活物（TA-KR，ON）（Lan等人， ，2014）（图1）。 TetR-KR或TA-KR分别结合TRE盒并在异源或真核细胞状态下诱导ROS损伤。如何染色质状态调节DNA损伤反应过程可以通过使用这种强大的方法来检查。
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