| RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of in vitro Transcribed mRNA in Human Whole Blood
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Author:
Date:
2020-12-20
[Abstract] The mRNA therapeutics is a new class of medicine to treat many various diseases. However, in vitro transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required. Here, we present a simple ex vivo method called ‘RNA ImmunoGenic Assay’ to measure immunogenicity of IVT-mRNAs in human whole blood. Chemically modified and unmodified mRNA are complexed with a transfection reagent (TransIT), and co-incubated in human whole blood. Specific cytokines are measured (TNF-α, INF-α, INF-γ, IL-6 and IL-12p70) using ELISAs. The ...
[摘要] [摘要] mRNA疗法是治疗多种疾病的新型药物。然而，我Ñ体外转录（IVT）的mRNA触发由于人类内体和细胞质RNA传感器，但是修饰的核苷的掺入识别的免疫应答已经示出吨ö减少此类反应。牛逼herefore ，测定标志着重要的环节人体免疫系统仍然需要。这里，我们提出一个简单的离体称为“RNA方法免疫ģ ENIC测定”测量的IVT-mRNA的免疫原性小号在人全血。将化学修饰和未修饰的mRNA与转染试剂（TransIT ）复合，并在人全血中共同孵育。特异性细胞因子测定（TNF- α ，INF- α ，INF- γ ，IL-6和IL-12p70的），使用的ELISA。进行qPCR分析以揭示特异性免疫途径的激活。所述RNA免疫ģ ENIC测定提供小号的简单且快速的方法来检测供体特异性-针对mRNA的治疗剂的免疫应答。
图形摘要：
RNA免疫基因测定的示意图
[背景] mRNA治疗是基因治疗的重要一类（Sahin等，2014 ；Antony等，2015 ...
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| Use of Optogenetic Amyloid-β to Monitor Protein Aggregation in Drosophila melanogaster, Danio rerio and Caenorhabditis elegans
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Author:
Date:
2020-12-05
[Abstract] Alzheimer’s Disease (AD) has long been associated with accumulation of extracellular amyloid plaques (Aβ) originating from the Amyloid Precursor Protein. Plaques have, however, been discovered in healthy individuals and not all AD brains show plaques, suggesting that extracellular Aβ aggregates may play a smaller role than anticipated. One limitation to studying Aβ peptide in vivo during disease progression is the inability to induce aggregation in a controlled manner. We developed an optogenetic method to induce Aβ aggregation and tested its biological influence in three model organisms–D. melanogaster, C. elegans and D. rerio. We generated a fluorescently labeled, optogenetic Aβ peptide that oligomerizes rapidly in vivo in the presence of blue light ...
[摘要] [摘要]Alzheimer'sdisease（AD）长期以来与淀粉样前体蛋白产生的细胞外淀粉样斑块（Aβ）的积聚有关。然而，在健康人身上发现了斑块，并不是所有的AD大脑都有斑块，这表明细胞外Aβ聚集体的作用可能比预期的要小。在疾病进展过程中研究Aβ肽的一个局限性是无法以可控的方式诱导聚集。我们开发了一种诱导Aβ聚集的光遗传学方法，并在三种模式生物中测试了其生物学效应：D.melanogaster、C.elegans和D.rerio。我们产生了一个荧光标记的，光生的
一种β肽，在所有生物体内，在蓝光存在下迅速寡聚。在这里，我们详细介绍了在动物模型中表达该融合蛋白的程序，使用延时光片显微镜研究对神经系统的影响，并进行代谢分析来测量由于细胞内Aβ聚集而引起的变化。这种方法利用光遗传学来研究AD的病理学，实现了目前任何其他方法都无法实现的体内时空控制。
[背景]阿尔茨海默病（AD）是一种衰弱的、与年龄相关的神经退行性疾病（Zhang等人，2011年；De ...
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| Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter
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Author:
Date:
2020-05-20
[Abstract] In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (Cas), have been developed in recent years. For the optimal use as well as continued developments of these genome editing tools, the evaluation of their efficiencies and accuracies is vital. Here, we present a protocol for a reporter based on frameshift fluorescence protein which we recently developed to evaluate the efficiency and accuracy of genome editing tools. In this method, a ~20 bp target sequence ...
[摘要] [摘要] 在过去的十年中，基因组编辑作为一种机制研究和潜在临床应用的新工具已成为关注的焦点。近年来，已开发出各种基因组编辑工具，例如大范围核酸酶，锌指核酸酶（ZFN），转录激活子样基于效应子的核酸酶（TALEN）以及成簇的规则间隔的短回文重复序列（CRISPR）相关基因（Cas）。 。对于这些基因组编辑工具的最佳使用和持续发展，评估其效率和准确性至关重要。在这里，我们介绍了一种基于移码荧光蛋白的报告子方案，我们最近开发了该方案以评估效率和 基因组编辑工具的实用性。在这种方法中，在天蓝色荧光蛋白（CFP）的起始密码子后插入一个约20 bp的包含移码的靶序列，以使其荧光失活，并且只有新的插入/缺失事件会重新激活CFP 荧光。 。为了增加可追溯性，将内部核糖体进入位点和红色荧光蛋白mCherryFP 放置在报告子的下游。由in / del介导的荧光恢复产生的CFP阳性细胞的百分比可以通过荧光测量装置定量，作为基因组编辑频率的读数。作为演示，我们在这里介绍CRISPR-Cas9技术的使用以及流式细胞仪作为荧光变化的读数。
[背景] 基因组编辑工具对于生物学机制的研究以及遗传疾病的预防和/或治疗非常重要（Maeder和Gersbach，2016）。在最近的几十年中，引入了几种基因组编辑工具，包括大范围核酸酶（Epinat 等，2003），锌指核酸酶（ZFN）（Kim ...
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