Name: spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease
Uploaded: 2012-02-03T13:00:00
Description: Watch this Scientific Journal Video about Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease at JoVE.com

作者想感谢他们的技术援助，希高桥布赖恩Muntean，邵螺，和布莱尔梅尔。支持这项工作是由美国国立卫生研究院（DK080640）和托莱多和ProMedica转化研究激励奖苏里亚Nauli博士大学的奖励。

1。细胞预处理和中期准备<ol><li>杜尔贝科的修改鹰的5％CO 2培养箱中10-15％的胎牛血清（FBS），1％青霉素/链霉素在37°C间（DMEM培养基）中的细胞生长，直到他们达到70-80％汇合。 </li><li>治疗细胞秋水仙胺溶液与0.05微克/毫升30-60分钟。 </li><li>任何浮动细胞中含有50毫升无菌猎鹰离心管收集介质。板sterile1X-PBS漂洗细胞。后用无菌胰蛋白酶孵育1-2分钟，收获和收集到同一管内的剩余细胞。 </...

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R., Matsui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+disruption+of+cytosine+DNA+methyltransferase+enzymes+induces+chromosomal+instability+in+human+cancer+cells.">Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells.</a> Cancer Research. 65, 8635-8639 (2005).</li><li>Matsui, S., Faitar, S. L., Rossi, M. R., Cowell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+spectral+karyotyping+to+the+analysis+of+the+human+chromosome+complement+of+interspecies+somatic+cell+hybrids.">Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids.</a> Cancer Genetics and Cytogenetics. 142, 30-35 (2003).</li><li>Nestor, A. L., Hollopeter, S. L., Matsui, S. 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光谱核型分析（天空）是一种细胞遗传学技术在研究基因和染色体组成。这种技术利用染色体涂染探针，这些探针的检测通过Sagnac干涉仪获得。完整的SKY过程通常需要大约一个星期，它涉及到几个关键步骤（ 图1）。天空采用了标准的协议，这是首次由帕迪拉纳什等 3。该协议已被适应各种细胞遗传学实验室，包括我们。 在协议中的关键步骤，质量...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液</td><td> cellgro敏达公司</td><td> 10-013-CV </td><td></td></tr><tr><td>胎牛血清（FBS）， </td><td>胎牛血清</td><td> SH30088-03 </td><td></td></tr><tr><td> penecillin /链霉素</td><td> Thermo Scientific的</td><td> SV30010 </td><td></td></tr><tr><td>秋水仙胺</td><td>罗氏诊断</td><td> 10 295 892 001 </td><td> 10微克/毫升</td></tr><tr><td>盐酸</td><td> Fisher Scientific则</td><td> A144-500 </td><td></td></tr><tr><td>氯化钾</td><td> Fisher Scientific则</td><td> S77375-1 </td><td></td></tr><tr><td>磷酸盐缓冲液</td><td> Thermo Scientific的</td><td> SH30256-01 </td><td></td></tr> &lt;TR&gt; <td>天空油漆探头套件（人力） </td><td>应用光谱成像</td><td> SKY000028 </td><td></td></tr><tr><td>天空油漆探头套件（鼠标） </td><td>应用光谱成像</td><td> SKY000030 </td><td></td></tr><tr><td>浓缩抗体检测试剂盒</td><td>应用光谱成像</td><td> SKY000033 </td><td></td></tr><tr><td>胰蛋白酶</td><td> Thermo Scientific的</td><td> SH30236.01 </td><td></td></tr><tr><td>甲醇</td><td> Fisher Scientific则</td><td> A433P-4 </td><td></td></tr><tr><td>醋酸</td><td> Fisher Scientific则</td><td> A38-212 </td><td></td></tr><tr><td> RNA酶A </td><td>罗氏诊断</td><td> 10 109 169 001 </td><td></td></tr><tr><td>胃蛋白酶</td><td> Sigma-Aldrich公司</td><td> P6887-5G </td><td></td></tr><tr><td> MgCl 2的 </td><td> Fluka公司</td><td> 63069-500ML </td><td></td></tr><tr><td> 37％的甲醛</td><td> mallinckrodt贝克</td><td> 2106-02 </td><td></td></tr><tr><td> 20X SSC的</td><td> Promega公司</td><td> V4261 </td><td></td></tr><tr><td>甲酰胺</td><td> Fluka公司</td><td> 47671 </td><td>准备只是在使用前</td></tr><tr><td>吐温20 </td><td> Fisher Scientific则</td><td> BP337-500 </td><td></td></tr><tr><td>显微镜的玻璃幻灯片</td><td> Fisher Scientific则</td><td> 12-549 </td><td></td></tr><tr><td>显微镜盖玻璃24x60mm </td><td>厂商VWR Vista的视觉</td><td> 16004-312 </td><td></td></tr><tr><td>橡皮泥</td><td>埃尔默（当地的零售商店） </td><td></td><td></td></tr><tr><td>杂交/ humidifiedchamber的/托盘</td><td> simport </td><td> M920-2 </td><td>在底部放湿纸巾</td></tr><tr><td>热基因扩增仪</td><td> Eppendorf公司</td><td> epgradient小号</td><td></td></tr><tr><td>振动平台/轨道摇床</td><td> bellco生物技术</td><td></td><td></td></tr><tr><td>振动/水浴</td><td>精密科学</td><td></td><td></td></tr><tr><td>的DAPI过滤立方体</td><td>色度技术</td><td></td><td></td></tr><tr><td>天空过滤立方体</td><td>色度技术</td><td></td><td></td></tr><tr><td> SpectraCube </td><td>应用光谱成像</td><td></td><td></td></tr><tr><td>倒置细胞培养显微镜</td><td>尼康</td><td>尼康的Eclipse TS100 </td><td></td></tr><tr><td>荧光显微镜</td><td>奥林巴斯</td><td> IX70 </td><td> 60X油</td></tr></tbody></table>

spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease

常规的方法来识别和分类 ​​，个别染色体取决于每个染色体的模式，在一个特定的物种正在分析1，2独特的带。然而，这种古典的带技术，是不可靠的识别复杂的，如那些与癌症相关的染色体畸变。为了克服带技术的局限性，光谱核型分析（SKY）介绍，提供许多染色体异常可靠的信息。 天空是多色荧光原位杂交（FISH）技术检测与中期染色体光谱显微镜3，4。天空已被证明是一个有价值的工具为一个范围广泛，大量的遗传性疾病和恶性肿瘤的5，6相关的染色体异常的细胞遗传学分析。天空涉及使用多色荧光标记的DNA探针准备从退化寡通过PCR otide引物。因此，每一个染色体有独特的光谱颜色后原位杂交探针，这些差异标记了荧光染料的混合物（罗丹明，得克萨斯红，Cy5标记，FITC标记和Cy5.5）。漫天的探头由55号染色体特异性探针7-10。 漫天的过程涉及几个步骤（ 图1）。天空要求高的有丝分裂指数从正常或病变的组织或血液细胞的可用性。从一个新鲜分离的原代细胞或细胞系单细胞的染色体玻片上传播。不同组合的每个染色体的特定荧光染料标记染色体蔓延。探头检测和图像采集，光谱成像系统由Sagnac干涉仪和CCD相机。这使得从样品发出的可见光光谱测量，获得光谱图像来回米个人染色体。 HiSKY，软件，用来拍摄的图像分析结果提供了一个易于识别的染色体异常。最终的结果是一个中期和核型分类的图像，每对染色体中有不同的颜色（ 图2）。这使得容易识别染色体的身份和易位。有关详细信息，请访问应用光谱成像网站（ <a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a> ）。 天空最近被用于鉴定染色体分离的缺陷和染色体异常在人类和小鼠的常染色体显性多囊肾病（多囊肾），一种遗传性疾病，特点是在初级纤毛功能障碍11-13。使用这种技术，我们证明了染色体分离异常和染色体缺陷的存在，在多囊肾患者和小鼠模型14。进一步分析使用的SKY不仅让我们能够找出染色体数目和身份，但也准确地检测出非常复杂的染色体畸变，如染色体缺失和易位（ 图2）。

Watch this Scientific Journal Video about Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease at JoVE.com

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

光谱核型分析（天空）是一种先进的细胞遗传学技术，以确定基因和染色体畸变。这种技术利用染色体涂染探针，这让所有染色体的分类。天空也可以识别复杂的染色体畸变，在小鼠和人类的各种疾病，包括多囊肾和种族隔离的缺陷。

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

科研

医学

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

5/6th联合切除术，高盐饮食和一氧化氮合酶抑制剂诱导Lewis大鼠慢性肾脏病

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

多囊肾一种可能的斑马鱼型号：击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

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 ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

适应人类Videofluoroscopic燕子研究的方法来检测和检定吞咽困难的小鼠疾病模型

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Surgical Models of Gastroesophageal Reflux with Mice

胃食管反流与小鼠手术模型

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

对称Bihemispheric死后的大脑切割学习健康和病理条件大脑在人

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

一个模型来模拟人类临床相关性缺氧

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...