光谱核型多囊肾病的研究在人类和小鼠的染色体异常
Summary
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。
Abstract
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（ http://www.spectral-imaging.com/ ）。
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）。
Protocol
1。细胞预处理和中期准备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。 治疗细胞秋水仙胺溶液与0.05微克/毫升30-60分钟。 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。 </…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the reagent | Company | Catalog number | Comments |
| DMEM | Cellgro Mediatech Inc. | 10-013-CV | |
| Fetal bovine serum (FBS) | Hyclone | SH30088-03 | |
| Penecillin/Streptomycin | Thermo Scientific | SV30010 | |
| Colcemid | Roche Diagnostics | 10 295 892 001 | 10 μg/ml |
| HCl | Fisher Scientific | A144-500 | |
| KCl | Fisher Scientific | S77375-1 | |
| Phosphate buffered saline | Thermo Scientific | SH30256-01 | |
| SKY paint probe kit (Human) | Applied Spectral Imaging | SKY000028 | |
| SKY paint probe kit (Mouse) | Applied Spectral Imaging | SKY000030 | |
| Concentrated antibody detection kit | Applied Spectral Imaging | SKY000033 | |
| Trypsin | Thermo Scientific | SH30236.01 | |
| Methanol | Fisher Scientific | A433P-4 | |
| Acetic acid | Fisher Scientific | A38-212 | |
| RNase A | Roche Diagnostics | 10 109 169 001 | |
| Pepsin | Sigma-Aldrich | P6887-5G | |
| MgCl2 | Fluka | 63069-500ML | |
| 37% Formaldehyde | Mallinckrodt Baker | 2106-02 | |
| 20X SSC | Promega | V4261 | |
| Formamide | Fluka | 47671 | prepare just before use |
| Tween-20 | Fisher Scientific | BP337-500 | |
| Microscope glass slides | Fisher Scientific | 12-549 | |
| Microscope cover glass 24x60mm | VWR Vista Vision | 16004-312 | |
| Rubber cement | Elmer’s (local retail store) | ||
| Hybridization/ humidifiedchamber/Tray | Simport | M920-2 | put wet paper towels at the bottom |
| Thermocycler | Eppendorf | Epgradient S | |
| Shaking platform/Orbital shaker | Bellco Biotechnology | ||
| Shaking/water bath | Precision Scientific | ||
| DAPI filter cube | Chroma Technology | ||
| SKY filter cube | Chroma Technology | ||
| SpectraCube | Applied Spectral Imaging | ||
| Inverted cell culture microscope | Nikon | Nikon Eclipse TS100 | |
| Fluorescence microscope | Olympus | IX70 | 60X oil |
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Tags
Spectral KaryotypingChromosome AbnormalitiesHumansMicePolycystic Kidney DiseaseBanding PatternClassical Banding TechniqueReliable IdentificationComplex Chromosomal AberrationsCancerSpectral Karyotyping (SKY)Multicolor Fluorescence In-situ Hybridization (FISH)Spectral MicroscopeCytogenetic AnalysisGenetic DiseasesMalignanciesMulticolor Fluorescently-labelled DNA ProbesDegenerate Oligonucleotide PrimersPCRUnique Spectral ColorIn-situ HybridizationFluorescent DyesRhodamineTexas RedCy5FITCCy5.5Chromosome Specific Probes
Citazione di questo articolo
AbouAlaiwi, W. A., Rodriguez, I., Nauli, S. M. Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease. J. Vis. Exp. (60), e3887, doi:10.3791/3887 (2012).