Name: chromosomics: detection of numerical and structural alterations in all 24 human chromosomes simultaneously using a novel octochrome fish assay
Uploaded: 2012-02-06T13:00:00
Description: Watch this Scientific Journal Video about Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay at JoVE.com

Forfatterne vil gerne takke Dr. Cliona M. McHale for kritisk læsning og redigering af manuskriptet. Dette arbejde blev finansieret af National Institute of Environmental Health Sciences, National Institute of Health tilskud R01ES017452 til L Zhang.

1. Stikprøve slide forberedelse <ul><li> OctoChrome FISH er designet til at undersoege kromosomale ændringer i metafasespredninger af humane celler, herunder men ikke begrænset til dyrkede perifere blodceller, stamceller / progenitorceller, og cellelinier. Prøver bør udarbejdes efter den normale procedure for høst metafaser 3,11-13: ved at ansætte colcemid behandling til at anholde celler i metafase, hypotonisk behandling til at svulme op celler, og Carnoys fixativ for at fastsætte celler i suspensi...

<ol>
	<li>Levsky, J. M., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization:+past,+present+and+future.">Fluorescence in situ hybridization: past, present and future.</a> J. Cell. Sci. 116, 2833-2838 (2003).</li><li>Zhang, L., Eastmond, D. A., Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+chromosomal+aberrations+detected+in+humans+exposed+to+benzene.">The nature of chromosomal aberrations detected in humans exposed to benzene.</a> Crit. Rev. Toxicol. 32, 1-42 (2002).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrations+in+chromosomes+associated+with+lymphoma+and+therapy-related+leukemia+in+benzene-exposed+workers.">Aberrations in chromosomes associated with lymphoma and therapy-related leukemia in benzene-exposed workers.</a> Environ. Mol. Mutagen. 48, 467-474 (2007).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzene+increases+aneuploidy+in+the+lymphocytes+of+exposed+workers:+a+comparison+of+data+obtained+by+fluorescence+in+situ+hybridization+in+interphase+and+metaphase.">Benzene increases aneuploidy in the lymphocytes of exposed workers: a comparison of data obtained by fluorescence in situ hybridization in interphase and metaphase.</a> Environ. Mol. Mutagen. 34, 260-268 (1999).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+aneusomy+and+long+arm+deletion+of+chromosomes+5+and+7+in+the+lymphocytes+of+Chinese+workers+exposed+to+benzene.">Increased aneusomy and long arm deletion of chromosomes 5 and 7 in the lymphocytes of Chinese workers exposed to benzene.</a> Carcinogenesis. 19, 1955-1961 (1998).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+translocations+and+aneusomy+in+chromosomes+8+and+21+among+workers+exposed+to+benzene.">Increased translocations and aneusomy in chromosomes 8 and 21 among workers exposed to benzene.</a> Cancer. Res. 58, 2176-2181 (1998).</li><li>Bayani, J., Squire, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+detection+of+chromosomal+aberrations+using+spectral+karyotyping.">Advances in the detection of chromosomal aberrations using spectral karyotyping.</a> Clin. Genet. 59, 65-73 (2001).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-wide+aneuploidy+study+(CWAS)+in+workers+exposed+to+an+established+leukemogen,+benzene.">Chromosome-wide aneuploidy study (CWAS) in workers exposed to an established leukemogen, benzene.</a> Carcinogenesis. 32, 605-612 (2011).</li><li>Rowley, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+chromosome+translocations+in+leukemogenesis.">The role of chromosome translocations in leukemogenesis.</a> Semin. Hematol. 36, 59-72 (1999).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+OctoChrome+fluorescence+in+situ+hybridization+to+detect+specific+aneuploidy+among+all+24+chromosomes+in+benzene-exposed+workers.">Use of OctoChrome fluorescence in situ hybridization to detect specific aneuploidy among all 24 chromosomes in benzene-exposed workers.</a> Chem. Biol. Interact. , 153-154 (2005).</li><li>Barch, M. J., Lawce, H. J., Arsham, M. S., Barch, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In The ACT cytogenetics laboratory. , 17-30 (1991).</li><li>Zhang, L., Yang, W., Hubbard, A. E., Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonrandom+aneuploidy+of+chromosomes+1,+5,+6,+7,+8,+9,+11,+12,+and+21+induced+by+the+benzene+metabolites+hydroquinone+and+benzenetriol.">Nonrandom aneuploidy of chromosomes 1, 5, 6, 7, 8, 9, 11, 12, and 21 induced by the benzene metabolites hydroquinone and benzenetriol.</a> Environ. Mol. Mutagen. 45, 388-396 (2005).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroquinone,+a+benzene+metabolite,+increases+the+level+of+aneusomy+of+chromosomes+7+and+8+in+human+CD34-positive+blood+progenitor+cells.">Hydroquinone, a benzene metabolite, increases the level of aneusomy of chromosomes 7 and 8 in human CD34-positive blood progenitor cells.</a> Carcinogenesis. 21, 1485-1490 (2000).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+benzene+health+effects+and+susceptibility.">Advances in understanding benzene health effects and susceptibility.</a> Annu. Rev. Public. Health. 31, 133-148 (2010).</li></ol>

Denne roman OctoChrome-FISH-analysen giver en samtidigt undersøge alle 24 menneskelige kromosomer i en enkelt hybridisering på et dias. Den arrangement af kromosom kombinationer på de 8 firkanter er blevet designet at lette identifikationen af ​​de ikke-tilfældige kromosom omlejringer i de mest almindelige leukæmier og lymfomer. Således kan analysen opdage numeriske (aneuploidi), såvel som strukturelle (translokationer) kromosom ændringer samtidigt. Det mest kritiske trin i dette...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer (valgfri) </td><tr><tr><td> OctoChrome Kit </td><td> Cytocell Ltd, Cambridge, England </td><td> PMP 803 </td><td></td></tr><tr><td> phytohaemagglutinin (PHA) </td><td> Invitrogen Corporation </td><td> 10576-015 </td><td></td></tr><tr><td> Colcemid </td><td> Invitrogen Corporation </td><td> 15212-012 </td><td></td></tr><tr><td> Carnoys fikseringsmiddel </td><td></td><td></td><td> Methanol: iseddikesyre = 3: 1 </td></tr><tr><td> Fluorescens mistativet </td><td></td><td></td><td> Udstyret med filtre for at se DAPI, Texas Red, FITC, og Coumarin spektre individuelt og en DAPI / FITC / Texas Red tredobbelt filter for at se forskellige farver på samme tid </td></tr><tr><td> Metafer software </td><td> MetaSystems, Altlußheim, Tyskland </td><td></td><td> Lette at finde alle metafaser og at re-evaluere de abnormiteter </td></tr></tbody></table>

chromosomics: detection of numerical and structural alterations in all 24 human chromosomes simultaneously using a novel octochrome fish assay

Fluorescens in situ hybridisering (FISH) er en teknik, der tillader specifikke DNA-sekvenser, der skal påvises på metafase eller interfase kromosomer i celle nuclei 1. Teknikken benytter DNA-sonder med unikke sekvenser, der hybridiserer til hele kromosomer eller specifikke kromosomale regioner, og fungerer som en kraftfuld supplement til klassiske cytogenetik. For eksempel rapporterede mange tidligere undersøgelser den hyppige påvisning af øget kromosomafvigelser i leukæmipatienter forbindelse med benzen eksponering, benzen-forgiftning patienter og raske arbejdstagere udsat for benzen, ved hjælp af klassiske cytogenetisk analyse 2. Ved hjælp af FISH, har leukæmi-specifikke kromosomale ændringer blevet observeret at være forhøjet i tilsyneladende raske arbejdstagere, der udsættes for benzen 3-6 med angivelse af de kritiske roller cytogentic ændringer i benzen-induceret leukemogenesis. Generelt en enkelt FISH analyse undersøger kun én eller nogle få hele kromosomereller specifikke loci per slide, så flere hybridiseringer skal udføres på flere dias til at dække alle de menneskelige kromosomer. Spectral karyotyping (SKY) giver mulighed for visualisering af hele genomet samtidigt, men kravet for speciel software og udstyr begrænser anvendelsen heraf 7. Her beskriver vi en ny FISH analyse, OctoChrome-fisk, som kan anvendes for Chromosomics, som vi definerer her som den samtidige analyse af alle de 24 menneskelige kromosomer på et dias i humane undersøgelser, såsom kromosom-dækkende aneuploidi undersøgelse (CWA-standarder) 8. Grundlag af den metoden, markedsføres af Cytocell som den Chromoprobe Multiprobe System, er en OctoChrome enhed, der er opdelt i 8 firkanter, som hver udoever tre forskellige helt kromosom maleri prober (figur 1). Hver af de tre proberne er direkte mærket med en forskellig farvet fluorofor, grøn (FITC), rød (Texas Red), og blå (Cumarin). Den arrangement af kromosom kombinationer på OctoChrome enheden har været designet til at lette identifikationen af ​​de ikke-tilfældige strukturelle kromosom ændringer (omplantninger) fundet i de mest almindelige leukæmier og lymfomer, for eksempel t (9; 22), t (15; 17), t (8; 21 ), t (14; 18) 9. Oevrigt, kan numeriske ændringer (aneuploidi) i kromosomer blive detekteret samtidigt. Den tilsvarende template Objektglasset også opdelt i 8 firkanter hvorpå metafase spreads er bundet (figur 2), og er placeret over den OctoChrome indretningen. Proberne og target DNA befinder denatureres ved høj-temperatur og hybridiseret i et fugtigt kammer, og derefter alle 24 humane kromosomer kan være visualiseres samtidigt. OctoChrome FISH er en lovende teknik til den kliniske diagnose af leukæmi og lymfom og for sporing af aneuploidies i alle kromosomer. Vi har anvendt denne nye Chromosomic fremgangsmåde i en CWA-standarder undersøgelse af benzen-eksponerede kinesiske arbejdere 8,10.

Watch this Scientific Journal Video about Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay at JoVE.com

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

En roman fluorescens<em&gt; In situ</em&gt; Hybridisering (FISH) metode, der simultant undersøger både numeriske og strukturelle kromosomanalyse ændringer, navnlig de specifikke kromosomale translokationer associeret med leukæmi og lymfom, af alle 24 humane kromosomer på en enkelt anordning i én hybridisering, er beskrevet.

Chromosomics: Påvisning af numeriske og strukturelle ændringer i alle 24 menneskelige kromosomer samtidig benytter en 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 ...

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