栉水母整片原位杂交。

CSH protocols Pub Date : 2008-11-01 DOI:10.1101/pdb.prot5087
Kevin Pang, Mark Q Martindale
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引用次数: 8

摘要

栉水母是一类海洋动物,其独特的生物学特征和系统发育位置使其成为理解动物进化的关键分类群。几乎所有栉水母都有一些特征,包括双向对称,由相连的纤毛组成的梳状排,一个顶端感觉器官和两个带有特殊粘附细胞的触手。迄今为止所研究的所有栉水母都有相同的刻板分裂程序,并经历一个特定的发育阶段,即cydippid幼虫,之后成虫结构发育并在物种之间产生很大差异;这对比较研究特别有用。在某些情况下,基因表达模式似乎是保守的。特别令人感兴趣的是,一些基因在栉水母体的区域中表达,这些区域在形态上与邻近区域没有区别。然而,一些基因的同源性很难确定,这可能是因为栉水母代表的极端分化。本方案描述了如何在栉水母胚胎和卵磷脂幼虫中固定、制备和杂交反义RNA探针,以及如何使用碱性磷酸酶偶联抗体和比色底物检测探针。利用这些技术,可以确定哪些细胞或组织表达感兴趣的基因。虽然该方案侧重于胚胎和幼虫样本,但该技术也可以应用于成体组织。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Ctenophore whole-mount in situ hybridization.

INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to fix, prepare, and hybridize antisense RNA probes in ctenophore embryos and cydippid larvae, as well as how to detect the probes using an alkaline phosphatase-conjugated antibody and colorimetric substrates. Using these techniques, it is possible to determine which cells or tissues express the gene of interest. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.

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