Name: spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease
Uploaded: 2012-02-03T13:00:00.000+00:00
Duration: 12 min 47 s
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。 </li><li> 1000 5分钟的转速旋转管，吸上清液留下0.5毫升，松开球，只用手指轻弹。 </li><li>根据颗粒大小，添加0.56％氯化钾DH 2低渗溶液5-10毫升，并在37°C孵育30-45分钟的暂停。 </li><li>添加一滴甲醇/醋酸（3:1​​体积/体积）每毫升低渗细胞悬液，轻轻颠倒混合管。 </li><li>在1200 rpm离心5分钟，收集沉淀步骤1.4，然后加入新鲜甲醇/醋酸（3:1体积/体积）固定液滴加5毫升，而轻弹颗粒continously。这个过程是关键，使中期利差不会被困在细胞的团块，这将危及实验。 </li><li>再次离心5分钟1200转，加5-10毫升的冰冷的甲醇/沿管壁醋酸固定液。在这个阶段，如果需要的话，这些细胞可以被保存在固定液收紧和短期或-80密封管在-20℃°C下长期（年）以供将来使用。 </li><li>无水乙醇清洁的幻灯片，然后浸在DH 2Ø为approximately10X的幻灯片，以形成滑动面水鞘。放置在玻璃板上和下降15-20μL细胞悬液的幻灯片（从1.8步放置在水浴在65-70°C间），10“以上的幻灯片。幻灯片为1-2分钟，晾干。 </li><li>干目标确保有中期染色体和利差是均匀分布的10X和40X光显微镜下检查的幻灯片。检查细胞质染色体周围存在。如果细胞质目前进行幻灯片预处理（胃蛋白酶消化），如果没有细胞质和染色体有良好的形态，那么就没有幻灯片预处理的需要。 </li></ol> 2。幻灯片预处理（胃蛋白酶处理） <ol><li>适用于120μL1:200酶溶液溶解到24毫米×60毫米显微镜盖玻片2X-SSC（20毫克/毫升）和中期反转滑面的玻璃罩上，然后轻轻地翻转中期滑面和孵育37°C为45分钟。 </li><li>小心地取出不刮伤幻灯片的玻璃罩和2X-SSC缓冲液洗在coplin壶每5分钟15分钟用颤抖的。 </li><li> 5-15μl蛋白酶库存解决方案的（DH 2 100毫克/毫升）加入到一个干净的烧杯中，然后加入100毫升预热（37℃）0.01M盐酸重要的是，胃被添加到一个干净的第一，不直接将盐酸溶液的烧杯中，否则它不会溶解在溶液中。孵育在coplin罐子含有盐酸/胃蛋白酶溶液，在37的幻灯片°C为3-5分钟。这一步是非常关键的太多消化会导致染色体overdigested太少消化细胞质将离开，未消化这可能会导致非特异性探针结合杂交信号的干扰。 </li><li>在coplin含5分钟，在室温下两次一罐100毫升1X-PBS洗幻灯片。 </li><li>洗的幻灯片，在coplin jar文件包含在室温下（1M MgCl 2的 50毫升，950毫升的1X-PBS）5分钟100毫升1X-PBS/MgCl 2。 </li><li>将在coplin罐子containin的幻灯片G 100毫升1％甲醛在室温（1X-PBS/MgCl 2 100毫升1.7毫升37％的甲醛）为10分钟。 </li><li>在coplin含5分钟一罐100毫升1X-PBS洗幻灯片。 </li><li>使用干40X镜头，以确保正常消化和幻灯片没有细胞质和染色体形态保留的光显微镜下观察的幻灯片。选择一个面积为杂交用金刚石笔。 </li></ol> 3。染色体和探针变性和杂交<ol><li>准备新鲜变性溶液（70％formamide/2X SSC，pH值7.0）和prewarm至70-80°C间，在coplin的jar包含在70水浴中变性的解决方案的coplin罐子放在水浴中放置在幻灯片°小鼠染色体和80 C度为30年代的1.5分钟的人类染色体。 </li><li>立即放置在冰冷的70％乙醇，80％的3分和100％的乙醇和空气干燥3分钟的幻灯片。检查幻灯片染色体的形态。好记黑暗染色体，而不是“相轻”或晕染色体染色体的形态。 </li><li>温暖的SkyPaint探头（天空油漆盒小瓶＃1）在37°C振荡20分钟，旋涡和离心机在1000 rpm短暂的几秒钟。 </li><li>在两个步骤的周期为5分钟周期由37℃60分钟允许为preannealing的标记的DNA探针在85°C编程1热循环变性探针。 </li><li> 10μL的变性探针应用到杂交和覆盖面积为22毫米×40毫米的玻璃罩确保不捕获的气泡。在37°C密封玻璃罩边缘用橡皮泥，在湿盒孵育48-72小时。 </li></ol> 4。荧光探针检测<ol><li>卸下玻璃罩仔细放置在一个coplin包含JAR的幻灯片预热（45℃）洗涤液（新鲜配制的50％2X SSC的甲酰胺）。洗5分钟45三次°C的振动水浴在45转</li><li>洗洗涤解决方案II（1X SSC）在45°C 5分钟两次用颤抖的幻灯片。 </li><li>解决方案三（4X SSC/0.1％吐温20）洗5分钟，45℃振荡Ç洗的幻灯片。 </li><li>阻断剂适用于80μL（天空油漆盒＃2小瓶），放置在37°C盖玻片孵育30分钟。 </li><li>删除幻灯片，让液体排出。适用于80μLCy5的染色试剂（浓缩抗体检测的CAD套件＃3小瓶）在37°C，适用于玻璃罩和孵育40分钟。 </li><li>洗洗涤溶液在45℃5分钟，用颤抖的第三三次幻灯片。 </li><li>适用于80μLCy5.5染色试剂（浓缩抗体检测的CAD套件＃4小瓶），放置在37°C玻璃罩和孵育40分钟。 </li><li>洗洗涤溶液在45℃5分钟，用颤抖的第三三次幻灯片。 </li><li>倾斜的幻灯片，并允许液体<html>流失。适用于20μL的DAPI的抗褪色试剂（天空油漆盒小瓶＃5），并放置一个24毫米×60毫米显微镜盖玻片。小心地取下空气泡沫可能已经形成。幻灯片可以立即成像，或保存于4°C，在不超过1周的黑暗。 </li></ol> 5。图像采集和分析<ol><li>通过查看中期幻灯片使用奥林巴斯显微镜配备了60X油浸没透镜，光谱立方体（定制设计的三带通滤波器），的DAPI过滤器与CCD相机Sagnac干涉仪模块，图像采集完成。 </li><li>光谱核型进行了使用后的天空视野软件（应用光谱成像版本1.62）的用户手册。 </li><li>染色体图像分析后，可以被看作（与特定的荧光颜色）的彩色图像，伪彩色图像（颜色分类）和倒置的DAPI图像（具体带型）。 </li></ol>e_title“&gt; 6代表结果。 一个完整的SKY过程通常需要一周左右的时间（ 图1）。这包括图像采集和分析提供中期细胞有充足的货源。染色体核型分析显示正常小鼠的染色体核型（40，XY）的野生型小鼠的细胞（ 图2a）。相比之下，PKD1细胞- / -小鼠（PKD的小鼠模型）显示在染色体数目和结构异常，如染色体缺失（8号染色体），易位（染色体11和19）（ 图2b），显着增加。我们还分析了从ADPKD患者的血管组织。一个简单计数染色体数目的研究表明，非多囊肾多囊肾血管样品，有23对染色体正常的数字（ 图2c）。然而，在一般情况下，我们观察到染色体分离失败，造成了46对染色体DPKD样本，而不是23对（ 图2d）。 <img alt="图1" src="/files/ftp_upload/3887/3887fig1.jpg" /> 图1。天空协议流程图。天空协议的流程图说明步骤来完成的实验，从细胞预处理和中期准备开始图像采集和分析。每一天一步一步的程序左侧大约一个星期的时间表。 <img alt="图2" src="/files/ftp_upload/3887/3887fig2.jpg" /> 图2。永生的小鼠细胞系和新鲜分离的人原代细胞的光谱核型分析。颜色和个别染色体倒置的DAPI图像显示之前的染色体排序。排序后，染色体是在“分类”表。提出了从野生型小鼠的中期蔓延）的天空图像包含染色体核型正常（40，XY）。 二） </st/ - -鼠标显示异常染色体数目（68 40）和染色体异常，如染色体缺失（8号染色体）和染色体易位（染色体11和19）ç）天荣&gt;从PKD1中期蔓延的天空图像从非多囊肾病人的血管组织的中期蔓延的形象，具有正常的核型（46，XX）。D）从多囊肾病人的血管组织的中期蔓延的天空图像包含的异常核型（92，XXXX的）。部分数据此前已报告和重用的许可14。

<ol>
	<li>Caspersson, T., Farber, S., Foley, G. E., Kudynowski, J., Modest, E. J., Simonsson, E., Wagh, U., Zech, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+differentiation+along+metaphase+chromosomes.">Chemical differentiation along metaphase chromosomes.</a> Experimental Cell Research. 49, 219-222 (1968).</li><li>Seabright, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+banding+technique+for+human+chromosomes.">A rapid banding technique for human chromosomes.</a> Lancet. 2, 971-972 (1971).</li><li>Padilla-Nash, H. M., Barenboim-Stapleton, L., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+analysis+of+human+and+mouse+chromosomes.">Spectral karyotyping analysis of human and mouse chromosomes.</a> Nature Protocols. 1, 3129-3142 (2006).</li><li>Schrock, E., Zschieschang, P., O'Brien, P., Helmrich, A., Hardt, T., Matthaei, A., Stout-Weider, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+of+human,+mouse,+rat+and+ape+chromosomes--applications+for+genetic+diagnostics+and+research.">Spectral karyotyping of human, mouse, rat and ape chromosomes--applications for genetic diagnostics and research.</a> Cytogenetic and Genome Research. 114, 199-221 (2006).</li><li>Padilla-Nash, H. M., Heselmeyer-Haddad, K., Wangsa, D., Zhang, H., Ghadimi, B. M., Macville, M., Augustus, M., Schrock, E., Hilgenfeld, E., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jumping+translocations+are+common+in+solid+tumor+cell+lines+and+result+in+recurrent+fusions+of+whole+chromosome+arms.">Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms.</a> Genes, Chromosomes &#38; Cancer. 30, 349-363 (2001).</li><li>Veldman, T., Vignon, C., Schrock, E., Rowley, J. D., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+chromosome+abnormalities+in+haematological+malignancies+detected+by+multicolour+spectral+karyotyping.">Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping.</a> Nature Genetics. 15, 406-410 (1997).</li><li>Karpf, A. 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. I., Allison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+genetic+complementation+controlling+the+chromosomal+abnormalities+and+loss+of+heterozygosity+formation+in+cancer.">A model for genetic complementation controlling the chromosomal abnormalities and loss of heterozygosity formation in cancer.</a> Cytogenetic and Genome Research. 116, 235-247 (2007).</li><li>Ried, T., Liyanage, M., du Manoir, S., Heselmeyer, K., Auer, K., Macville, M., Schrock, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cytogenetics+revisited:+comparative+genomic+hybridization+and+spectral+karyotyping.">Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping.</a> Journal of Molecular Medicine (Berlin, Germany). 75, 801-814 (1997).</li><li>AbouAlaiwi, W. A., Takahashi, M., Mell, B. R., Jones, T. J., Ratnam, S., Kolb, R. J., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+polycystin-2+is+a+mechanosensitive+calcium+channel+involved+in+nitric+oxide+signaling+cascades.">Ciliary polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades.</a> Circulation Research. 104, 860-869 (2009).</li><li>Nauli, S. M., Alenghat, F. J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A. E., Lu, W., Brown, E. M., Quinn, S. J., Ingber, D. E., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystins+1+and+2+mediate+mechanosensation+in+the+primary+cilium+of+kidney+cells.">Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.</a> Nature Genetics. 33, 129-137 (2003).</li><li>Nauli, S. M., Kawanabe, Y., Kaminski, J. J., Pearce, W. J., Ingber, D. E., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cilia+are+fluid+shear+sensors+that+regulate+calcium+signaling+and+nitric+oxide+production+through+polycystin-1.">Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1.</a> Circulation. 117, 1161-1171 (2008).</li><li>AbouAlaiwi, W. A., Ratnam, S., Booth, R. L., Shah, J. V., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+from+humans+and+mice+with+polycystic+kidney+disease+are+characterized+by+polyploidy+and+chromosome+segregation+defects+through+survivin+down-regulation.">Endothelial cells from humans and mice with polycystic kidney disease are characterized by polyploidy and chromosome segregation defects through survivin down-regulation.</a> Human Molecular Genetics. 20, 354-367 (2011).</li><li>Padilla-Nash, H. M., Nash, W. G., Padilla, G. M., Roberson, K. M., Robertson, C. N., Macville, M., Schrock, E., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cytogenetic+analysis+of+the+bladder+carcinoma+cell+line+BK-10+by+spectral+karyotyping.">Molecular cytogenetic analysis of the bladder carcinoma cell line BK-10 by spectral karyotyping.</a> Genes, Chromosomes &#38; Cancer. 25, 53-59 (1999).</li></ol>

没有利益冲突的声明。

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，质量好的中期的准备是非常重要的，有一个成功的SKY分析。例如，如果染色体有太多的细胞质或如果幻灯片是太旧，图像质量可能会受到影响。作为影片剪辑中我们看到，在细胞质中的内容可以很容易地确定使用的高清晰度差干涉对比显微镜技术。此外，积极分裂细胞的可用性是漫天分析的先决条件，这可以通过与秋水仙胺治疗更长的时间或其他生长因子的细胞除了克服。天空的整体成功率在很大程度上取决于用户的技能和经验。 天空提供了染色体分析的强大和可靠的工具，包括在一个单一的染色体结构的变化。在除了蜂窝多倍体，天空提供了可能性，以研究染色体插入，删除，复制和易位。例如，天空允许新颖和隐藏的染色体畸变的鉴定和识别复杂的重排在癌症的发生和发展15。此外，天空被应用于一个研究领域内的比较细胞遗传学研究4在进化过程中的染色体重排。使用的SKY技术我们的实验室成为第一个在小鼠和人类多囊肾病（ 图2），以确定染色体异常。毫无疑问，天空将有利于在许多其他染色体相关疾病。事实上，我们建议，天空也将是有益的许多纤毛相关的发病机制，其中纤毛已被证明是调节细胞分裂14。 由于小鼠和大鼠是重要的动物模型来研究疾病的机理，并在体内进行功能评估，小鼠和大鼠的SKY分析探头的发展，已允许例行的大鼠和小鼠染色体核型。目前，天空因此仅限于在人类，大鼠和小鼠的染色体研究。然而，随着越来越多的研究实验室使用其他物种的研究基因组的组成，它是可预见的更多不同的生物体染色体涂染探针将很快市售。

<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 ...

spectral-karyotyping-to-study-chromosome-abnormalities-humans-mice

Nanyang Technological University

Research

JoVE Journal

Medicine

38.3K 次观看.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. 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。

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

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

科研

遗传学

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

免疫与感染

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

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

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

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

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 ...

医学

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

生物学

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.

使用3D机器人超 声进行小鼠 肾脏体内分析

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), ...

Cytogenetics

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell&#8217;s chromosomal number and structure, together known as its karyotype. Many chromosomal abnormalities are associated with disease. Each chromosome in a karyotype can be stained with a variety of dyes to give unique banding patterns. More recent techniques, including comparative genomic hybridization and fluorescence in situ hybridization (FISH), allow for detecting specific chromosomal features or abnormalities.This video will begin by examining the principles of these classical and modern cytogenetics techniques. This is followed by an examination of a general protocol for performing FISH. Finally, several examples of how karyotyping can be applied to various medical applications are presented.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

摘要

探索更多视频

此视频中的章节

通过多色荧光跨染色体畸变稳定的检测

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

在多囊肾小型啮齿动物模型采用超高场磁共振成像

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

染色体制备培养细胞

斑马鱼肾功能评价采用荧光测定间隙

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

使用3D机器人超声进行小鼠肾脏体内分析

细胞遗传学

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

摘要

探索更多视频

此视频中的章节

通过多色荧光跨染色体畸变稳定的检测

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

在多囊肾小型啮齿动物模型采用超高场磁共振成像

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

染色体制备培养细胞

斑马鱼肾功能评价采用荧光测定间隙

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

使用3D机器人超 声进行小鼠 肾脏体内分析

细胞遗传学

使用3D机器人超声进行小鼠肾脏体内分析