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

Авторы хотели бы поблагодарить Брайана Мунтян, Шао вот, Маки Такахаши и Блэр Мелл за техническую помощь. Эта работа была поддержана награды от NIH (DK080640) и Университет Толедо и ProMedica Поступательное премии стимулирование исследований доктора Сурья Наули.

1. Предварительная обработка клеток и метафазных подготовки <ol><li> Клетки, выращенные в модификации Дульбекко среднего орла (DMEM), содержащих 10-15% эмбриональной телячьей сыворотки (FBS) и 1% пенициллина / стрептомицина при 37 ° С с 5% СО 2 инкубатора, пока они не достигают 70-80% слияния. </li><li>...

<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>

Спектральный Кариотипирование (SKY) является цитогенетики техника, используемая в изучении генома и хромосомные композиций. Эта техника использует датчики хромосомы живописи, и обнаружение этих зондов, полученных в результате Саньяка интерферометра. Весь процесс SKY обычно занимает ок?...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии </td></tr><tr><td> DMEM </td><td> CellGro Mediatech Инк </td><td> 10-013-CV </td><td></td></tr><tr><td> Эмбриональной телячьей сыворотки (FBS) </td><td> Hyclone </td><td> SH30088-03 </td><td></td></tr><tr><td> Penecillin / стрептомицином </td><td> Thermo Scientific </td><td> SV30010 </td><td></td></tr><tr><td> Колцемид </td><td> Roche Diagnostics </td><td> 10 295 892 001 </td><td> 10 мкг / мл </td></tr><tr><td> HCl </td><td> Fisher Scientific </td><td> A144-500 </td><td></td></tr><tr><td> KCl </td><td> Fisher Scientific </td><td> S77375-1 </td><td></td></tr><tr><td> Фосфатного буфера </td><td> Thermo Scientific </td><td> SH30256-01 </td><td></td></tr> &lt;TR&gt; <td> SKY краски зонд комплект (Human) </td><td> Прикладные спектральных изображений </td><td> SKY000028 </td><td></td></tr><tr><td> SKY краски зонда набор (мышь) </td><td> Прикладные спектральных изображений </td><td> SKY000030 </td><td></td></tr><tr><td> Концентрированный комплект обнаружения антител </td><td> Прикладные спектральных изображений </td><td> SKY000033 </td><td></td></tr><tr><td> Трипсин </td><td> Thermo Scientific </td><td> SH30236.01 </td><td></td></tr><tr><td> Метанол </td><td> Fisher Scientific </td><td> A433P-4 </td><td></td></tr><tr><td> Уксусная кислота </td><td> Fisher Scientific </td><td> A38-212 </td><td></td></tr><tr><td> РНКазы </td><td> Roche Diagnostics </td><td> 10 109 169 001 </td><td></td></tr><tr><td> Пепсин </td><td> Sigma-Aldrich </td><td> P6887-5G </td><td></td></tr><tr><td> MgCl 2 </td><td> Fluka </td><td> 63069-500ML </td><td></td></tr><tr><td> 37% формальдегида </td><td> Mallinckrodt Бейкер </td><td> 2106-02 </td><td></td></tr><tr><td> 20-кратный SSC </td><td> Promega </td><td> V4261 </td><td></td></tr><tr><td> Формамид </td><td> Fluka </td><td> 47671 </td><td> готовить непосредственно перед употреблением </td></tr><tr><td> Твин-20 </td><td> Fisher Scientific </td><td> BP337-500 </td><td></td></tr><tr><td> Слайды стекло микроскопа </td><td> Fisher Scientific </td><td> 12-549 </td><td></td></tr><tr><td> Микроскоп покровного стекла 24x60mm </td><td> VWR Vista, Видение </td><td> 16004-312 </td><td></td></tr><tr><td> Резиновый цемент </td><td> Элмер (местный розничный магазин) </td><td></td><td></td></tr><tr><td> Гибридизация / humidifiedchamber / лоток </td><td> Simport </td><td> M920-2 </td><td> положить мокрые полотенца бумаги на дне </td></tr><tr><td> Thermoвелосипедист </td><td> Эппендорф </td><td> Epgradient S </td><td></td></tr><tr><td> Тряска платформы / Орбитальный шейкер </td><td> Bellco биотехнологии </td><td></td><td></td></tr><tr><td> Тряска / водяной бане </td><td> Точность научного </td><td></td><td></td></tr><tr><td> DAPI фильтр куб </td><td> Chroma технологии </td><td></td><td></td></tr><tr><td> SKY фильтр куб </td><td> Chroma технологии </td><td></td><td></td></tr><tr><td> SpectraCube </td><td> Прикладные спектральных изображений </td><td></td><td></td></tr><tr><td> Инвертированный микроскоп культуре клеток </td><td> Nikon </td><td> Nikon Eclipse TS100 </td><td></td></tr><tr><td> Флуоресцентный микроскоп </td><td> Олимп </td><td> IX70 </td><td> 60X нефти </td></tr></tbody></table>

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

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 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

Спектральный Кариотипирование (SKY) является передовой техникой цитогенетики для идентификации геномных и хромосомных аберраций. Эта техника использует датчики хромосомы картины, которые позволяют классификации всех хромосом. SKY можно также определить комплекс хромосомных аберраций и разделение дефектов у мышей и людей с различными заболеваниями, в том числе поликистоз почек.

Спектральный Кариотипирование по изучению хромосомных аномалий у человека и мыши с поликистозом почек

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

Ортотопической трансплантации аорты у мышей для изучения сосудистых заболеваний

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

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 нефрэктомии в сочетании с высоким содержанием соли и азотной ингибирование синтазы, чтобы побудить хронического заболевания почек у крыс Льюиса

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

Оценка функции сосудов у больных с хронической болезнью почек

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

Возможно данио рерио Модель поликистоза почек: Нокдаун<em&gt; Wnt5a</em&gt; Причины кисты у рыбок данио почек

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

Адаптация человека Videofluoroscopic Ласточка изучит методы обнаруживать и определять характеристики Дисфагия в мышиных Disease Models

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

Использование аденоассоциированные Касперского как инструмент для изучения сетчатки барьеров при болезни

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

Использование ультра-высокой полевой МРТ малого грызунов Модели поликистоз почек для<em&gt; В Vivo</em&gt; Фенотипирование и медикаментов мониторинга

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

Хирургические Модели гастроэзофагеальной рефлюксной с мышами

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

Симметричный Bihemispheric Посмертное мозга резки для изучения здоровые и патологически Мозговые условия у людей

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

Модель Моделировать клинически значимых гипоксию в организме человека

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