高解析度全山<em>在原位</em杂交斑马鱼胚胎内，研究基因表达和功能
Summary
在脊椎动物的发育和疾病的基因功能研究的模型，小热带鱼，斑马鱼已成为一种流行。靶基因表达的时间和空间可以由<em在原位</em>杂交。我们改进后的协议允许低非特异性背景信号检测低丰度的成绩单。
Abstract
本文着重介绍整个安装的斑马鱼胚胎原位杂交（WISH）。无论是在组织中的分布和发育阶段基因表达的愿望技术有助于评估。议定书描述使用WISH斑马鱼胚胎，使用地高辛标记的反义RNA探针。通过在体外转录的基因已被克隆和线性化的模板，将地高辛联核苷酸探针所产生的。收获定义的发育阶段的胚胎绒毛膜特异性探针孵化前被删除。经过洗涤步骤，以除去多余的探针，胚胎用碱性磷酸酶偶联的抗地高辛抗体孵育。通过采用碱性磷酸酶的显色底物，特定基因的表达进行评估。根据不同的基因的表达水平，就可以完成整个过程需要2-3天。
Introduction
斑马鱼（ 斑马鱼 ）已经成为一个强大的动物模型，为研究脊椎动物的发育，疾病，行为，以及在药物筛选1-3。斑马鱼的胚胎，可以得到大量从一个单一的十字路口。测土配方施肥和发展发生子宫外和透明的胚胎发育迅速。关键发育事件发生在第一受精后48小时（HPF）。这包括器官原基的外观和细胞分化的启动。击倒或过度表达的蛋白质可以通过在吗啉代反义寡核苷酸（MO）胚胎的显微注射，或上面的一个或两个细胞阶段（0.75 HPF）的分别的mRNA。
斑马鱼胚胎的愿望，有利于利益的特定基因的时空表达的研究。后的愿望技术应用显微注射胚胎mRNA或MO过表达式ESS或击倒特定的蛋白质水平揭示微分调节其他基因。
可以是相关的基因表达模式的变化与表型的变化，在器官发生早期揭示的靶基因的功能。由于探头可以预先制备和贮存，原位杂交技术可以应用在一个时间使用六孔板和定制的篮子，以显示至少20个基因的基因表达模式。
Protocol
1。整个安装原位杂交斑马鱼胚胎 1.1胚胎的制备收集胚胎发育阶段所需。请参阅基梅尔等 5萌芽阶段的描述。 删除手动绒毛膜使用钳（杜蒙列表镊子5号）。 修复胚胎在4％的多聚甲醛（PFA）的PBS中的溶液（磷酸盐缓冲盐水），在4℃下过夜胚胎用PBS洗净，每次5分钟，在室温下至少3倍。 商店胚胎在-20°C?…
Representative Results
每筐50个胚胎（每个基因/实验条件），该基因的表达模式，可以实现在一个实验中使用的协议。几乎所有的胚胎显示一个特定的基因表达模式相似。 原位杂交染色的代表性实例示于图3-6中 。 的正义和反义核糖探针，合成的cDNA对应于PC5.1，PC5.2 6，SCL/tal-1 7，GATA-1 8,9，FLK / kdrl的10，11嘘12 DLX2的fkd7 / FOXA1 13，14,15，胰岛素和胰?…
Discussion
我们已经开发了改进的方法具有高的分辨率的可视化RNA。的原位杂交程序，进行了使用简单的量身定制筐与多孔底的（图1）。胚胎的处理，在无菌条件下，在6孔培养板中，在室温下使用RNase-free的解决方案。
是由不同颜色的Eppendorf管中，以分隔胚胎筐。筐带或不带的轮辋是用于容易取向，并确定为对应于一个特定的探针的购物篮内的胚胎。
<p class="jove_c…Disclosures
The authors have nothing to disclose.
Materials
| Taq Polymerase | Invitrogen | 10342053 | |
| TopoTA Cloning Kit- Dual Promoter pCRII-TOPO | Invitrogen | K4650-01 | |
| HQ Mini Plasmid Purification Kit | Invitrogen | K2100-01 | |
| Agarose | Roche | 11 685 660 001 | |
| Not1 and HindIII Restriction Enzymes | Invitrogen | 15441-025, 15207-012 | |
| Buffer Saturated Phenol, ultrapure | Invitrogen | 15513039 | Store at 4 °C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling. |
| Chloroform | Fisher | C607-1 | Toxic and suspected carcinogen. Work under fume hood. |
| RNase-free DNAse I | Roche | 4716728001 | Prepare small aliquots and store at -20°C. |
| DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase | Roche | 11277073910 | |
| RNase Inhibitor | Roche | 10777-019 | |
| 3.0 M Sodium Acetate (pH 5.5) | Fisher | 50-751-7355 | |
| 100% Ethanol | Commercial alcohols | ||
| Diethyl Pyrocarbonate (DEPC) | Sigma | 40718-25ML | DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask. |
| NaOH | Fisher | SS255-1 | |
| EDTA 0.5 M (pH 8.0) | Fisher | 50-751-7404 | |
| Instant Ocean Salt | Aquarium Systems | N/A | |
| Phosphate Buffered Saline (PBS) | Fisher | FL-03-0900 | |
| Paraformaldehyde (PFA) | Sigma | P6148-1KG | Prepare and store at -20°C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask. |
| Phenylthiocarbomide (PTC) | Sigma | P7629-25G | PTC is highly toxic. Use safety glasses, gloves, and dust mask. |
| Methanol | Fisher | A947-4 | |
| Dumont (Watchmaker’s) Forceps pattern no. 5 | Fine Science Tools | ||
| Six-well Cell Culture Cluster | Sarstedt | 83.1839 | |
| Baskets are made of nylon mesh and Eppendorf tubes | In-house preparation | To make the baskets, cut Eppendorf tubes (1.5 ml) with or without rims to remove the conical end. Cut nylon mesh into small pieces corresponding to the size of the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical hot plate until both the tube and nylon mesh stick together (carry out in fume hood). Cut off the excess mesh from the baskets and store them in 100% methanol until used. | |
| Polyoxyethylenesorbitan Monolaurate (Tween 20) | Sigma | P1379-500ML | |
| Proteinase K | Fermentas | EO0491 | |
| Acetic Anhydride | Sigma | 242845 | |
| Triethanolamine | Sigma | 90279-100ML | |
| Formamide, high purity grade | Sigma | F9037 | |
| AG501-X8 Resin | Bio Rad | 142-6424 | |
| Citric Acid monohydrate | Sigma | C0706-500G | |
| Heparin Sodium Salt | Sigma | H3393-25KU | |
| Saline-sodium Citrate Buffer (SSC) | Sigma | S6639 | |
| tRNA from bakers yeast type X | Sigma | R5636-1ML | |
| NaCl | Fisher | S640-500 | |
| Tris-HCl | Fisher | BP153-1 | |
| MgCl2 | Fisher | S25403 | |
| Levamisole Hydrochloride | Sigma | 31742 | |
| N,N-Dimethylformamide anhydrous (DMF) |
Sigma | 227056 | Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles. |
| 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) | Sigma | N6639 | Protect this solution from light. |
| Nitroblue tetrazolium (NBT) | Sigma | 840W | Protect this solution from light. |
| Albumin from Bovine Serum, purified fraction V (BSA) | Sigma | A8806 | |
| Sheep Anti-digoxigenin-AP Fab Fragments | Roche | 1093274910 | |
| Glycerol | Sigma | G5516-500ML | |
| 96-well cell culture plates | Sarstedt | 83.1835 | |
| Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2 | ZIRC | Tg(mnx1:GFP)ml2 | |
| Instant Ocean | Aquarium Systems | N/A |
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Cite This Article
Chitramuthu, B. P., Bennett, H. P. J. High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function. J. Vis. Exp. (80), e50644, doi:10.3791/50644 (2013).