| A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy
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Author:
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2021-04-05
[Abstract] The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis (Chang et al., 2020). This protocol was used in the 3D ...
[摘要] [摘要]自由生活的线虫秀丽隐杆线虫是研究发育生物学的流行模型系统。在这里，我们描述了详细的协议，以高压冷冻线虫的胚胎（解剖后离体，或完整的蠕虫内），然后快速冷冻替代。经过处理的样品适合通过常规电子显微镜（EM）或更新的体积EM（vEM）方法（如Focuse d离子束扫描电子显微镜（FIB-SEM））进行超微结构分析。的细胞特征，例如超微结构的NUCL耳信封，染色体，内质网和线粒体保存良好这些实验程序后，并产生精确的三维模型用于可视化和分析（张等人，2020）。在秀丽隐杆线虫合子的前核相遇后，该方案被用于膜和染色体的3D重建（Rahman等，2020）。
[背景技术]线虫是自由生活线虫具有许多特性，使其适合于科学的研究：（1）将蠕虫是〜1毫米长; ...
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| Detection and Quantification of African Swine Fever Virus in MA-104 Cells
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Author:
Date:
2021-03-20
[Abstract] Detection of live African swine fever virus (ASFV) has historically relied on the use of primary swine macrophages (PSM). PSM do not replicate and have to be isolated fresh from donor swine. We previously identified that a MA-104 cells (ATCC #CRL-2378.1), a commercially available cell line isolated from African green monkey (Cercopithecus aethiops) kidney epithelial cells, supports the detection of ASFV from field samples with a sensitivity comparable to that of primary swine macrophages. Collection of swine blood or lungs is time costing, which is often not readily available in most veterinary diagnostic laboratories. MA-104 cells could thus be used as substitute for primary swine macrophages to save significant lead time by avoiding the production of primary swine macrophages. ...
[摘要] [摘要]活的非洲猪瘟病毒（ASFV）的检测在历史上一直依赖于原代猪巨噬细胞（PSM）的使用。PSM不能复制，必须从供体猪中新鲜分离。我们先前发现，MA-104细胞（ATCC＃CRL-2378.1）是一种从非洲绿猴（Cercopithecus aethiops ）肾上皮细胞分离的商业细胞系，支持从野外样品中检测ASFV，其灵敏度可与ASFV媲美。原发性猪巨噬细胞。Ç的猪的血液或肺ollection是时间成本计算，这往往是在大多数兽医诊断实验室容易获得的。MA-104细胞因此可以用作原代猪巨噬细胞的替代品，通过避免原代猪巨噬细胞的产生来节省大量的准备时间。
[背景]非洲猪瘟病毒（ASFV）的成员，非洲猪瘟病毒科的家庭，导致野猪和家猪具有高度传染性和致命性出血热，即非洲猪瘟（ASF）。成熟的病毒颗粒（病毒粒子）直径为175-215 nm，脂质双层包裹了二十面体衣壳和180-190千碱基对的双链DNA基因组。根据宿主特征和病毒株，该病毒会引起多种症状，包括高度致死性至亚临床性（Tulman等，2009 ...
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| Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus
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Author:
Date:
2020-09-05
[Abstract] The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus.
We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows ...
[摘要] [摘要 ] 单独基于CRISPR靶序列仍难以预测单个CRISPR / Cas9-sgRNA的切割效率。不同的细胞内环境（例如，取决于细胞类型或细胞周期状态）可能会通过DNA修饰（例如表观遗传标记和DNA结合蛋白（例如组蛋白））和染色质状态的改变来改变基因组DNA的可及性，从而影响sgRNA效率。核内基因组DNA的标记。
我们recen TLY 报道的多步骤方法筛选用于有效sgRNAs靶向的识别的单纯疱疹病毒（HSV-1）的基因组，并报告为在潜对裂解病毒相抑制CRISPR-Cas9的差动机构。该协议中详述的筛选平台允许在无细胞系统中以及在病毒靶细胞（例如人包皮成纤维细胞）的背景下逐步测试切割效率，然后对CRISPR / sgRNA的作用进行功能测试病毒蛋白的表达，复制，和再活化。该策略可以容易地应用于其他靶细胞，例如多能干细胞衍生的人感觉神经元或其他人DNA病毒。
背景 ] 单纯疱疹病毒（HSV）是的一种嗜神经性DNA病毒疱疹病毒科家族引起终身和无法治愈感染在大多数人群的，并可能导致显著发病率和死亡率（Liesegang环等人，1989; Roizman 等人。，2013） ...
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