Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes
Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes
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DOI：
10.3791/3709
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发表时间：
2012-06-01
影响因子：
1.2
通讯作者：
James, Anthony A.
中科院分区：
文献类型：
--
作者：
Juhn, Jennifer;James, Anthony A.
蚊子是一系列病原体的媒介，包括虫媒病毒、原生动物寄生虫和线虫。研究蚊子宿主和病原体相互作用的组织以及参与繁殖的器官中表达的转录本和基因调控因子，对于减少蚊媒疾病传播和扰乱卵子发育的策略具有重要意义。许多工具已经被用来研究和验证基因表达的时间和组织特异性调节。在这里，我们描述了已经开发的用于获得空间信息的协议，该协议增强了我们对特定基因表达及其产物积累的理解。所描述的方法已用于验证转录本在与蚊媒病原体传播相关的组织中的表达和积累模式，如女性唾液腺，以及与蚊子繁殖和发育有关的卵巢和胚胎的亚细胞室。以下程序代表了一种优化的方法，在不损失靶标特异性杂交信号的情况下提高了协议中各个步骤的效率。A部分介绍了RNA探针制备、软组织解剖以及固定和杂交的一般程序，而B部分详细介绍了蚊子胚胎的收集、固定、预杂交和杂交的具体步骤。
Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development.The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.