| Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection
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Author:
Date:
2017-10-05
[Abstract] Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny.
[摘要] 显微注射是线虫秀丽隐杆线虫遗传转化中最常用的工具，促进基因的转基因表达，通过聚集的定期散布的短回文重复序列（CRISPR）-Cas9系统的基因组编辑或转录 dsRNA用于RNA干扰（RNAi）。 外源DNA被递送到成年雌雄同株的种系中的发育中的卵母细胞中，然后在它们的后代中产生转基因动物。 在该方案中，我们描述了显微注射程序和随后的转基因后代选择。
【背景】在C.通过显微注射的DNA转化通常用于产生过表达或异位表达可以与标签（例如，绿色荧光蛋白[GFP]）融合的基因的转基因动物，允许突变体的表型拯救和/或蛋白质的定位和功能的分析（Carter等人，1990; Chalfie等人，1994; Mello和Fire，1995）。聚集的定期散布的短回文重复（CRISPR）-Cas9系统的出现需要显微注射以通过引入点突变或插入/缺失突变来实现高度特异性的基因组编辑（概述于Dickinson和Goldstein，2016）。此外，该技术被应用于dsRNA的可诱导和/或组织特异性转录以便于遗传性RNA干扰（RNAi）（Tavernarakis等人，2000） ...
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| Chemotaxis and Jumping Assays in Nematodes
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Author:
Date:
2015-09-20
[Abstract] Nematodes have sensitive olfactory perception, which is used to detect and differentiate many volatile odorants. Some odorants are attractive, others repulsive, and yet others evoke no particular response. Chemotaxis assays can be used to determine the role of certain odors in many different behaviors including foraging, predator avoidance, and mate attraction. In addition to chemotaxis, some species of nematodes in the entomopathogenic genus Steinernema can jump, which is thought to play an important role in host-seeking and dispersal (Dillman and Sternberg, 2012). Jumping and chemotaxis assays have been successfully used to identify odorants that stimulate these behaviors in a variety of nematodes (Bargmann et al., 1993; Campbell and Kaya, 1999; Hallem et al., ...
[摘要] 线虫具有敏感的嗅觉感知，其用于检测和区分许多挥发性气味剂。 一些气味有吸引力，其他排斥，还有一些没有引起特别的反应。 趋化性测定可用于确定某些气味在许多不同行为中的作用，包括觅食，捕食者回避和配偶吸引。 除了趋化性之外，昆虫病原属Steinernema中的一些种类的线虫可以跳跃，这被认为在宿主寻找和分散中起重要作用（Dillman和Sternberg，2012）。 跳跃和趋化性测定已经成功地用于鉴定在多种线虫中刺激这些行为的气味剂（Bargmann等人，1993; Campbell和Kaya，1999; Hallem等人， 2011; Dillman等人，2012; Castelletto 等人，2014）。 这里基于文献的成长体提供了趋化性和跳跃测定的详细方案。
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| Post-crystallization Improvement of RNA Crystals by Synergistic Ion Exchange and Dehydration
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Author:
Date:
2015-09-05
[Abstract] Compared to the recent dramatic growth in the numbers of genome-wide and functional studies of complex non-coding RNAs, mechanistic and structural analyses have lagged behind. A major technical bottleneck in the structural determination of large RNAs and their complexes is preparation of diffracting crystals. Empirically, a vast majority of such RNA crystals fail to diffract X-rays to usable resolution (~4 Å) due to their inherent disorder and non-specific packing within the crystals. Here, we present a protocol that combines post-crystallization cation replacement and dehydration that dramatically improved the diffraction quality of crystals of a large gene-regulatory mRNA-tRNA complex. This procedure not only extended the resolution limit of X-ray data from 8.5 to 3.2 Å, but also ...
[摘要] 与最近在复杂的非编码RNA的基因组范围和功能研究的数量的显着增长相比，机械和结构分析已经落后。在大RNA及其复合物的结构确定中的主要技术瓶颈是衍射晶体的制备。经验上，由于其固有的无序和在晶体内的非特异性堆积，绝大多数这样的RNA晶体不能将X射线衍射到可用的分辨率（〜4）。在这里，我们提出一个协议，结合后结晶阳离子替代和脱水，大大改善晶体的大基因调节mRNA-tRNA复杂的衍射质量。该程序不仅将X射线数据的分辨率极限从8.5扩展到3.2，而且还显着提高了数据的质量，实现了重新定相和结构确定。因为它利用了反离子和溶剂化在RNA结构中的一般重要性，这个程序可能证明在其他大型非编码RNA的晶体学分析中广泛有用。
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