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

Forfatterne vil gerne takke Brian Muntean, Shao Lo, Maki Takahashi og Blair Mell for deres tekniske assistance. Dette arbejde blev støttet af priser fra NIH (DK080640) og The University of Toledo &amp; ProMedica Translationel Forskning Stimulation Award til Dr. Surya Nauli.

1. Celle præbehandling og metafase forberedelse <ol><li> Cellerne dyrkes i Dulbeccos Modifikation af Eagle &#39;s Medium (DMEM) indeholdende 10-15% føtalt bovint serum (FBS) og 1% penicillin / streptomycin ved 37 ° C med 5% CO 2 inkubator, indtil de når 70-80% konfluens. </li><li> Behandle de cellerne med colcemid opløsning ved 0,05 ug / ml for 30-60 min. </li><li> Indsamle den medium indeholdende eventuelle flydende celler i 50-ml sterile falk at centrifugerør. Skyl cellerne på pladen med sterile1X-...

<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>

Spectral karyotyping (SKY) er en cytogenetik teknik, der anvendes i at studere genomiske og kromosom-kompositioner. Denne teknik drager fordel af kromosom maleri prober, og påvisning af disse prober er erhvervet gennem en sagnac interferometer. Den komplette SKY proces tager normalt omkring en uge, og det indebærer flere vigtige skridt (figur 1). Det SKY bruger en standard protokol, som første gang blev beskrevet af Padilla-Nash et al 3. Protokollen er siden blevet tilpasset af fo...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nr. </td><td> Kommentarer </td></tr><tr><td> DMEM </td><td> Cellgro Mediatech Inc. </td><td> 10 til 013-CV </td><td></td></tr><tr><td> Føtalt bovint serum (FBS) </td><td> Hyclone </td><td> SH30088-03 </td><td></td></tr><tr><td> Penecillin / Streptomycin </td><td> Thermo Scientific </td><td> SV30010 </td><td></td></tr><tr><td> Colcemid </td><td> Roche Diagnostics </td><td> 10 295 892 001 </td><td> 10 ug / ml </td></tr><tr><td> HCI </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> Phosphatbufret saltvand </td><td> Thermo Scientific </td><td> SH30256-01 </td><td></td></tr> &lt;tr&gt; <td> SKY maling probe kit (Human) </td><td> Anvendt Spectral Imaging </td><td> SKY000028 </td><td></td></tr><tr><td> SKY maling probe kit (Mouse) </td><td> Anvendt Spectral Imaging </td><td> SKY000030 </td><td></td></tr><tr><td> Koncentreret antistof påvisning kit </td><td> Anvendt Spectral Imaging </td><td> SKY000033 </td><td></td></tr><tr><td> Trypsin </td><td> Thermo Scientific </td><td> SH30236.01 </td><td></td></tr><tr><td> Methanol </td><td> Fisher Scientific </td><td> A433P-4 </td><td></td></tr><tr><td> Eddikesyre </td><td> Fisher Scientific </td><td> A38-212 </td><td></td></tr><tr><td> RNase A </td><td> Roche Diagnostics </td><td> 10 109 169 001 </td><td></td></tr><tr><td> Pepsin </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% Formaldehyd </td><td> Mallinckrodt Baker </td><td> 2106-02 </td><td></td></tr><tr><td> 20X SSC </td><td> Promega </td><td> V4261 </td><td></td></tr><tr><td> Formamid </td><td> Fluka </td><td> 47671 </td><td> forberede lige før brug </td></tr><tr><td> Tween-20 </td><td> Fisher Scientific </td><td> BP337-500 </td><td></td></tr><tr><td> Mikroskop glas objektglas </td><td> Fisher Scientific </td><td> 12-549 </td><td></td></tr><tr><td> Mikroskop cover glas 24x60mm </td><td> VWR Vista Vision </td><td> 16004-312 </td><td></td></tr><tr><td> Gummi cement </td><td> Elmer &#39;s (lokale butik) </td><td></td><td></td></tr><tr><td> Hybridisering / humidifiedchamber / Tray </td><td> Simport </td><td> M920-2 </td><td> satte våde papirservietter nederst </td></tr><tr><td> Thermocycler </td><td> Eppendorf </td><td> Epgradient S </td><td></td></tr><tr><td> Rystebord / Orbital shaker </td><td> Bellco Bioteknologi </td><td></td><td></td></tr><tr><td> Ryste / vandbad </td><td> Precision Scientific </td><td></td><td></td></tr><tr><td> DAPI-filter terning </td><td> Chroma Technology </td><td></td><td></td></tr><tr><td> SKY filter terning </td><td> Chroma Technology </td><td></td><td></td></tr><tr><td> SpectraCube </td><td> Anvendt Spectral Imaging </td><td></td><td></td></tr><tr><td> Inverteret cellekultur mikroskop </td><td> Nikon </td><td> Nikon Eclipse TS100 </td><td></td></tr><tr><td> Fluorescens mikroskop </td><td> Olympus </td><td> IX70 </td><td> 60X olie </td></tr></tbody></table>

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

Konventionel metode at identificere og klassificere individuelle kromosomer afhænger af den unikke banding mønster af hvert kromosom i en specifikke arter, der analyserede 1, 2. Denne klassiske banding teknik, imidlertid, er ikke er pålidelig i at identificere komplekse kromosomforandringer såsom dem associeret med cancer. For at overvinde de begrænsninger af banding teknik, er Spectral karyotyping (SKY), indført til at give meget pålidelige oplysninger om kromosomfejl. SKY er en flerfarve fluorescens in-situ hybridisering (FISH) teknik til at detektere metafasekromosomer med spektral mikroskop 3, 4. SKY har vist sig at være et værdifuldt værktøj til cytogenetisk analyse af en bred vifte af kromosom abnormiteter i forbindelse med et stort antal af genetiske sygdomme og maligniteter 5, 6. SKY involverer anvendelsen af ​​flerfarvede fluorescens-mærkede DNA-prober fremstillet ud fra den degenererede oligonucleotide primere ved PCR. Således, hver kromosom har et unikt spektral farve efter in-situ hybridisering med prober, der er differentielt mærket med en blanding af fluorescerende farvestoffer (Rhodamin, Texas Red, Cy5, FITC og Cy5.5). Proberne, der anvendes for SKY bestå af op til 55 kromosomområder specifikke prober 7-10. Den Proceduren for SKY involverer flere trin (figur 1). SKY kræver tilgængeligheden af ​​celler med højt mitotisk indeks fra normalt eller sygt væv eller blod. Kromosomerne af en enkelt celle fra enten en frisk isoleret primær celle eller en cellelinie er spredt på et objektglas. Dette kromosom spread er mærket med en forskellig kombination af fluorescerende farvestoffer specifikke for hvert kromosom. Til detektering og image erhvervelse består spektral imaging system sagnac interferometer og et CCD-kamera. Dette tillader måling af det synlige lysspektrum udsendt fra prøven og til at erhverve en spektral billede tilbagem. individuelle kromosomer. HiSKY, den software, der bruges til at analysere resultaterne af de billeder, giver det let at identificere kromosom anomalier. Slutresultatet er en metafase, og en karyotype klassifikation billede, i hvilken hvert par af kromosomer har en distinkt farve (figur 2). Dette giver mulighed for nem identifikation af kromosom identiteter og omplantning. For flere detaljer, kan du besøge Applied Spectral Imaging hjemmeside ( <a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a> ). SKY blev for nylig anvendt til en identifikation af kromosom adskillelse fejl og kromosomfejl hos mennesker og mus med autosomal dominant polycystisk nyresygdom (ADPKD), en genetisk sygdom karakteriseret ved dysfunktion i primære cilier 11-13. Ved at bruge denne teknik, vi viste tilstedeværelsen af unormal kromosom segregering og kromosomfejl i ADPKD patienter og musemodeller 14. Yderligere analyser anvender SKY ikke kun lov for os at identificere kromosomalt antal og identitet, men også nøjagtigt at detektere meget komplekse kromosomforandringer såsom kromosomområder deletioner og omplantning (figur 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

Spectral karyotyping (SKY) er en avanceret cytogenetik teknik til at identificere genomiske og kromosomforandringer. Denne teknik drager fordel af kromosom maleri prober, hvilket muliggøre klassificering af alle kromosomer. SKY kan også identificere komplekse kromosomafvigelser og adskillelse mangler i mus og mennesker med forskellige sygdomme, herunder polycystisk nyresygdom.

Spectral karyotyping til Studieinformationen kromosomfejl hos mennesker og mus med polycystisk nyresygdom

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

Ortotopisk Aorta Transplantation i mus for Studiet af Vascular Disease

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

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

Vurdering af vaskulære funktion hos patienter med kronisk nyresygdom

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

En mulig Zebrafisk Model af polycystisk nyresygdom: Knockdown af<em&gt; Wnt5a</em&gt; Årsager cyster i zebrafisk Nyrer

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

Tilpasse det menneskelige Videofluoroscopic Swallow Study metoder til påvisning og karakterisere Dysfagi i murine sygdomsmodeller

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

Brug Adenoassocieret virus som værktøj til at studere Retinale Barrierer i Disease

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

Anvendelse af Ultra-high Field MRI i Small gnavermodeller af polycystisk nyresygdom for<em&gt; In vivo</em&gt; Fænotypebestemmelse 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 ...

Surgical Models of Gastroesophageal Reflux with Mice

Kirurgiske modeller af gastroøsofageal refluks med Mus

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

Symmetric Bihemispheric Postmortem Brain Skæring at studere Sunde og Patologisk Brain Betingelser i mennesker

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

En model til simulere klinisk relevant Hypoxi i mennesker

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