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秀丽隐杆线虫基本实验方法:同步化与观察

Basic Caenorhabditis elegans methods: synchronization and observation.

作者信息

Porta-de-la-Riva Montserrat, Fontrodona Laura, Villanueva Alberto, Cerón Julián

机构信息

Department of Cancer and Human Molecular Genetics, Bellvitge Institute for Biomedical Research.

出版信息

J Vis Exp. 2012 Jun 10(64):e4019. doi: 10.3791/4019.

Abstract

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved. C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology, aging, stem cell biology and germ line biology. An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

摘要

对线虫秀丽隐杆线虫的分子与发育生物学研究始于20世纪70年代初,由悉尼·布伦纳发起,此后它被广泛用作模式生物。秀丽隐杆线虫具有简单、透明和生命周期短等关键特性,这些特性使其多年来一直是基础生物学研究的合适实验系统。在这种线虫中的发现具有广泛影响,因为许多控制动物发育的细胞和分子过程在进化上是保守的。秀丽隐杆线虫的生命周期经历一个胚胎阶段和四个幼虫阶段,然后才发育为成虫。发育时间根据温度不同可能需要2至4天。在每个阶段都可以观察到几个特征性性状。其完整的细胞谱系知识以及对其基因组的深入注释,使这种线虫成为神经生物学、衰老、干细胞生物学和生殖系生物学等众多领域的优秀模式。秀丽隐杆线虫成为一个有吸引力的研究模式的另一个特点是,通过相对简单的方案就有可能获得处于特定阶段同步化的虫群。易于饲养和繁殖这种线虫,再加上同步化的可能性,提供了一个强大的工具来获得大量的线虫,可用于各种小型或高通量实验,如RNA干扰筛选、微阵列、大规模测序、免疫印迹或原位杂交等。由于其透明性,使用微分干涉相差显微镜(也称为诺马斯基显微镜)可以在显微镜下区分秀丽隐杆线虫的结构。例如,使用荧光DNA结合剂4',6-二脒基-2-苯基吲哚(DAPI),可以特异性识别和定位单个细胞,以及与其相关的亚细胞结构/缺陷。

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