| A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy
|
|
Author:
Date:
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; ...
|
|
|
| 3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation
|
|
Author:
Date:
2018-06-05
[Abstract] Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, ...
[摘要] 细胞在固体表面生长为单层的传统二维细胞培养仍然代表了药物检测癌症研究的标准方法。然而，在2D培养物中生长的细胞缺乏相关的细胞基质和细胞 - 细胞相互作用，并且忽略实体肿瘤的真实三维解剖结构。在2D中培养的细胞也可经历细胞骨架重排并获得与异常基因表达相关的人造极性（Edmondson等人，2014）。最近开发出更好地模拟体内情况的3D文化系统。用于研究癌症干细胞的3D体外肿瘤模型（肿瘤球体）在该领域已经获得了越来越多的普及（Weiswald等人，2015）。使用基质嵌入或封装的球体，悬滴培养的球体，磁悬浮系统或3D打印方法的系统已经广泛用于研究和新药筛选。在本文中，我们描述了测试shRNA介导的基因沉默对肿瘤球体形成和生长的影响的详细方案。这种方法允许研究人员测试基因敲低对肿瘤起始细胞生长的影响。正如我们实验室所证实的那样，该方案也可用于直接从肿瘤组织中分离3D癌细胞系。
【背景】3D体外肿瘤细胞模型代表了细胞系与体内生长的肿瘤之间的桥接实验方法（Pampaloni等人，2007; ...
|
|
|
| Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
|
|
Author:
Date:
2018-04-05
[Abstract] For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones.
[摘要] 对于疾病和基础科学研究而言，功能丧失（LOF）突变是非常重要的。 在这里，我们提供了一个简单的流线化协议来产生LOF iPSC系列，通过将CRISPR载体与附加型重编程质粒同时引入成纤维细胞，规避了传统基因编辑和已建立的iPSC系的克隆的技术挑战。 我们的实验已经产生了常染色体基因中所有3种基因型的几乎偶数。 此外，我们提供了一个详细的方法来维护和iPSC克隆的96孔板的基因分型。
【背景】CRISPR / Cas9技术允许简单且特异地针对特定基因组位置进行基因编辑。将该技术与诱导性多能干细胞（iPSC）的疾病建模和再生医学潜力相结合将继续对生物医学研究产生前所未有的影响。然而，使CRISPR / Cas9系统适应iPSC已经提出了几个挑战。在细胞系中进行基因编辑的传统方法是用表达Cas9蛋白质的质粒和指导RNA（gRNA）转染细胞，然后产生单克隆并筛选所需的遗传改变。不幸的是，iPSC不适用于单细胞克隆。已经开发了几种补充媒介和克隆方法来克服这一困难，但仍然充满昂贵的设备（低氧培养箱），困难的技术步骤（FACS分选的单个iPSC的存活）或劳动密集型方案（亚克隆）（Forsyth ，2006; Miyaoka ...
|
|
|