多囊肾一种可能的斑马鱼型号：击倒<em> Wnt5a的</em>原因在斑马鱼肾囊肿
Summary
We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.
Abstract
Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG – and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.
Introduction
斑马鱼（ 斑马鱼 ）胚胎已被广泛用作模型为研究肾发展和多囊肾病。有许多优势，使用斑马鱼作为动物模型：研究遗传相互作用的可行性，使用反义吗啉（MO），用于蛋白质敲低的能力，有机会迅速测定大量的胚胎，并且容易观察器官的表型活幼虫1。该前肾是第一个肾在脊椎动物发展和功能是在幼虫斑马鱼2。斑马鱼前肾的结构相对简单相比哺乳动物后肾，第三和最后肾发展的哺乳动物。肾单位是肾脏的工作单元，其中每个人肾脏500,000-1,000,000肾3,4和具有约13000肾5各小鼠肾脏之间含有，因此很难观察单肾结构我ñ人或小鼠肾脏。斑马鱼只有两个肾单位，每个斑马鱼肾包含所有在肾小球和小鼠和人类6和类似专门肾细胞类型的小管中发现的主要组件。相比其他脊椎动物模型如爪蟾 ，斑马鱼肾更接近于哺乳动物肾，因为它有一个封闭的系统7。
在最近几年中，斑马鱼的基因组已经被测序，允许广泛介绍的遗传工具，广泛突变体资源，并在斑马鱼的模型转基因报告线路集合。 12-72小时受精后（HPF）之间的斑马鱼前肾表格可在透明的胚胎很容易被可视化。在肾母细胞瘤蛋白WT1是肾脏发育的一个重要因素。所述wt1b启动子的Tg的控制下表达绿色荧光蛋白（GFP）转基因斑马鱼线（wt1b：GFP）显示GFP expre裂变专门设在pronephric地区的斑马鱼胚胎，从17 HPF 8起。 Nephronophthisis（NPHP），一种常染色体隐性囊性肾脏疾病，是由NPHP基因9的突变引起。 NPHP4被击倒吗啉导致囊肿形成的的Tg（wt1b：GFP）鱼10因此，这种转基因鱼是一个合适的模型，观察肾脏结构和囊肿形成肾脏发育过程中。重要的是，肾脏发育的调节剂的影响，可以利用该菌株中一个时间和劳动高效方式进行研究。
我们的论文描述了使用的Tg（wt1b：GFP）鱼作为模式基因调制后显现肾囊肿形成。我们使用的开始和剪切位点反义的MO打掉了Wnt5a基因斑马鱼。而Wnt5a是Wnt信号家族的非规范的分泌糖蛋白，发挥各器官和细胞产后的发展具有重要作用功能11。 Wnt5a的工作原理是通过非经典Wnt途径，包括平面细胞极性（PCP）途径，已经发现肾小管伸长时发挥导向细胞分裂的作用。 Wnt5a的通过形成与受体样酪氨酸激酶（RYK），其中，还转导Wnt5a的信令通过形成与VANGL平面细胞极性蛋白2（Vangl2）的复合物，从而促进Vangl2稳定性12复杂调节的Wnt / PCP通路。在PCP途径缺陷可能导致随机的细胞分裂，并导致肾囊肿形成。我们使用的Tg（wt1b：GFP）斑马鱼线以下的Wnt5a敲除，观察肾囊肿形成。 的Tg（wt1b：GFP）斑马鱼模型允许实时成像，及时观察肾脏结构。 Wnt5a的击倒后，肾囊肿形成被发现开始，在24 HPF;在72 HPF，囊肿可能在肾小球和近端小管中找到。这种方法也可用于为screen其他基因可能导致肾囊肿形成。
Protocol
Representative Results
Discussion
多囊肾（PKD）是终末期肾脏疾病的主要原因在人类中之一，其特征在于进行性囊肿形成，肾肿大，与异常小管发育14。常染色体显性多囊肾（ADPKD）是一种遗传性疾病，其中突变要么PKD1的，编码多囊蛋白1（PC1），或PKD2，多囊编码-2（PC2），导致多囊性肾脏。许多其它基因，特别是那些编码在初级纤毛中发现的蛋白质，被认为是参与肾囊肿的发展，至今尚无理想的工具来筛选这些基因。我?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是支持由美国国立卫生研究院（DK093625到LH和DK069909和DK047757至JHL）和VA（优异奖I01BX000820到JHL）。我们要感谢博士迈克尔·包和何洁博士在宾夕法尼亚斑马鱼核心的大学提供必要的支持。
Materials
| 0.5% Phenol-Red | Sigma | P0290 | Color indicator for injection |
| Morpholino | Genn Tools, LLC | (customized) | Customized designed to the gene of interest |
| Pre-pulled needle | Tritech Research | MINJ-PP | If large amount of needle is required, you can also purchase a needle puller and prepare the needle in the lab |
| T7 mMessage Kit | Ambion | 1344 | For in vitro transcription to make capped mRNA for rescue experiment |
| N-Phenylthiourea | Sigma | P7629 | For prevention of melanization |
| Tricaine | Sigma | A5040 | Also called ethyl 3-aminobenzoate, for zebrafish anesthesia |
| Methyl Cellulose | Sigma | M-0387 | For position of zebrafish |
| QIAquick PCR Purification Kit | Qiagen | 28104 | For purification of PCR product for cap RNA synthesis. |
| dNTP mix | Promega | U1511 | For PCR |
| Tag DNA Polymerase | Invitrogen | 10342-053 | For PCR |
| Equipment | |||
| NanoDrop sepctrophotometer | Thermo Scientific | ND-1000 | For measure morpholino and RNA concentration |
| Air Compressor | Werther International, Inc. | Panther Compact 106 | Air source for injection |
| Pico micro-injection pump | World Precision Instruments Inc | PV830 Pnematic PicoPump | Other types of microinjection system can be used. |
| Micro-manuplator | World Precision Instruments Inc | MMJR | Right-handed (MMJL for left handed) |
| Needle holder | World Precision Instruments Inc | 5430-ALL | To hold needle for micromanipulation |
| Dumont Tweezers | Fine Surgical Tools | 11253-20 | For breaking off the needle tip |
| Dissecting microscope | Leica M205C | For observing and imaging zebrafish embryos | |
| Fluorscence microscope | Zeiss Axio Obserer D1m | For imaging zebrafish pronephros | |
| Capillary tube (I.D 0.15 mm) | VitroCom | CV1525Q-100 | For measure the volume of each injected drop |
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