| Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures
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Author:
Date:
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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| Preparation and Immunofluorescence Staining of the Trachea in Drosophila Larvae and Pupae
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Author:
Date:
2016-05-05
[Abstract] The Drosophila melanogaster trachea is a branched network of rigid chitin-lined tubes that ramify throughout the body and functions as the fly’s respiratory organ. Small openings at the ends of the tracheal tubes allow gas exchange to occur by diffusion between internal tissues and the exterior environment. Tracheal tubes are lined by a single layer of epithelial cells, which secrete chitin and control tube morphology and size. Studies of tracheal development in Drosophila embryos have elucidated fundamental mechanisms of tube morphogenesis and maintenance in vivo, and identified major signaling pathways that regulate these processes (Manning and Krasnow, 1993; Affolter and Shilo, 2000; Zuo et al., 2013; Kerman et al., ...
[摘要] melanogaster 气管是一种由几丁质内衬的管组成的分支网络，分布在整个身体并作为苍蝇的呼吸器官。气管管端部的小开口允许通过内部组织和外部环境之间的扩散而发生气体交换。气管管由单层上皮细胞衬里，其分泌几丁质和对照管形态和尺寸。在Drosop hila 胚胎中的气管发育的研究阐明了管形态发生和维持体内的基本机制，并且鉴定了调节这些过程的主要信号传导途径（Manning和Krasnow，1993; Affolter和Shilo，2000; Zuo等人，2013; Kerman等人，2006; Schottenfeld等人， em，2010）。近年来，在变态过程中对气管的兴趣日益增长，当在幼虫中用作呼吸器官的气管分支被修复或被由致动的气管祖细胞产生的新的气管组织或成熟的气管细胞（Manning和Krasnow，1993; Sato和Kornberg，2002; Guha等人，2008; Guha和Kornberg，2005; Weaver和Krasnow，2008; Pitsouli和Perrimon， 2010; Chen和Krasnow，2014）在过程结束时形成成人气管。正在进行的衰变和组织形成模拟其他生物体中组织修复和再生的方面，并且已经用于理解祖细胞如何分裂和分化（Pitsouli和Perrimon，2010; ...
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| Chick Neural Tube Explant Culture
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Author:
Date:
2015-10-05
[Abstract] The neural tube explant culture technique allows in vitro culturing of small pieces of neural tissue isolated from e.g., chick or mouse embryonic tissue in a matrix of collagen for defined periods of time. This method can be used to study the effects of defined molecules on developmental processes such as neural progenitor proliferation and neuronal differentiation and/or survival. Since the explant material can also be prepared from embryonic tissue electroporated with expression vectors, this technique can be adapted to study gene function in the presence of specific environmental signals. Different regions of the neural tube can also be isolated during the dissection step, allowing specific regions of the neural tube to be studied separately. Here, we present a neural ...
[摘要] 神经管外植体培养技术允许在胶原基质中从例如小鸡或小鼠胚胎组织分离的小片神经组织的体外培养定义的时间段。 该方法可用于研究限定分子对发育过程如神经祖细胞增殖和神经元分化和/或存活的影响。 由于外植体材料也可以从用表达载体电穿孔的胚胎组织制备，该技术可以适于在特定环境信号的存在下研究基因功能。 也可以在解剖步骤期间分离神经管的不同区域，允许单独研究神经管的特定区域。 在这里，我们提出了我们已经在几个研究中使用的神经管外植体培养方法（Dias等人，2014; Lek等人，2010; Vallstedt， et al。，2005）。
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