Quiring Rebecca, Wittbrodt Beate, Henrich Thorsten, Ramialison Mirana, Burgtorf Carola, Lehrach Hans, Wittbrodt Joachim
Developmental Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany.
Mech Dev. 2004 Jul;121(7-8):971-6. doi: 10.1016/j.mod.2004.03.031.
Gene expression profiling is an important component of functional genomics. We present a time and cost efficient high-throughput whole-mount in situ technique to perform a large-scale gene expression analysis in medaka fish (Oryzias latipes) embryos. Medaka is a model system ideally suited for the study of molecular genetics of vertebrate development. Random cDNA clones from an arrayed stage 20 medaka plasmid library were analyzed by whole-mount in situ hybridization on embryos of three representative stages of medaka development. cDNA inserts were colony PCR amplified in a 384-format. The PCR products were used to generate over 2000 antisense RNA digoxigenin probes in a high-throughput process. Whole-mount in situ hybridization was carried out in a robot and a broad range of expression patterns was observed. Partial cDNA sequences and expression patterns were documented with BLAST results, cluster analysis, images and descriptions, respectively; collectively this information was entered into a web-based database, "MEPD" (http://www.embl-heidelberg.de/mepd/), that is publicly accessible.
基因表达谱分析是功能基因组学的重要组成部分。我们提出了一种省时且经济高效的高通量整体原位技术，用于在青鳉鱼（Oryzias latipes）胚胎中进行大规模基因表达分析。青鳉是一个理想的模型系统，非常适合用于研究脊椎动物发育的分子遗传学。从一个排列好的20期青鳉质粒文库中随机选取的cDNA克隆，通过对青鳉发育三个代表性阶段的胚胎进行整体原位杂交来分析。cDNA插入片段在384孔板中进行菌落PCR扩增。PCR产物用于在高通量过程中生成2000多个地高辛标记的反义RNA探针。整体原位杂交在机器人中进行，并观察到了广泛的表达模式。部分cDNA序列和表达模式分别通过BLAST结果、聚类分析、图像和描述进行记录；这些信息共同被录入一个基于网络的数据库“MEPD”（http://www.embl-heidelberg.de/mepd/），该数据库可公开访问。
