Khila Abderrahman, Abouheif Ehab
Department of Biology, McGill University, Montréal, Québec, Canada H3A 1B1.
Cold Spring Harb Protoc. 2009 Jul;2009(7):pdb.prot5250. doi: 10.1101/pdb.prot5250.
The detection of transcript distribution throughout a fixed tissue is a major step in studying the transcriptional activity of target genes and their function. In situ hybridization specifically detects the spatial distribution of RNA transcripts using an antisense RNA probe. This protocol describes the preparation of digoxigenin-labeled antisense RNA probes and their hybridization to complementary mRNA sequences; expression can then be localized using an antibody against digoxigenin conjugated to a chromogenic enzyme. In ants, this method can be applied to visualize cell populations of interest among other populations in a tissue, such as insect ovaries, or in the whole organism, such as insect embryos. Specific markers (e.g., genes known to be expressed in particular clusters of cells) can be cloned and used as probes to study the distribution and development of germline cells (e.g., nanos, vasa), neurons (repo), or limb structures (e.g., distal-less). Various markers might also allow the study of oogenesis (nanos, par-1, oskar), segmentation in the embryo (e.g., engrailed, wingless), or other developmental processes.
检测固定组织中整个转录本的分布是研究靶基因转录活性及其功能的重要步骤。原位杂交使用反义RNA探针特异性检测RNA转录本的空间分布。本方案描述了地高辛标记的反义RNA探针的制备及其与互补mRNA序列的杂交；然后可以使用与显色酶偶联的抗地高辛抗体定位表达。在蚂蚁中，这种方法可用于在组织中的其他细胞群体（如昆虫卵巢）或整个生物体（如昆虫胚胎）中可视化感兴趣的细胞群体。可以克隆特定标记（例如已知在特定细胞簇中表达的基因）并用作探针来研究生殖细胞（例如nanos、vasa）、神经元（repo）或肢体结构（例如distal-less）的分布和发育。各种标记还可能有助于研究卵子发生（nanos、par-1、oskar）、胚胎中的节段化（例如engrailed、wingless）或其他发育过程。
