Name: chromosome preparation from cultured cells
Uploaded: 2014-01-28T13:00:00
Description: Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Arbejdet er beskrevet i dette manuskript blev muliggjort af midler fra Patrick F. Taylor Foundation.

1.. Kromosom Høst af adhærerende celler <ol><li> Standard protokol <ol><li> Grow cellerne overensstemmelse med specifikke celledyrkningsbetingelser. Når cellerne har nået logaritmisk fase (80% konfluens), tilsættes 10 ul / ml Colcemid til cellekulturen kolben. Et minimum på 2 x 10 6 celler anbefales. </li><li> Cellerne inkuberes ved 37 ° C i en 5% CO2-inkubator i 45 min. Ved hjælp af en steril pipette medier fra celler i en 15 ml konisk rør. Braklægning. </li><li> Vask ...

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Vi har udnyttet den foreliggende procedure for kromosom isoleret fra celler af forskellige organismer, herunder forskellige celletyper opnået fra human, makakaber, rotter og mus 11,20-22. Den standard-protokol er forudsat, men visse vigtige skridt og variabler skal muligvis justeres for disse forskellige typer af celler. Flere specifikke trin er afgørende i både forberedelsen kromosom-og G-banding at sikre den bedst mulige kvalitet af resultaterne. En af de variabler, som kan påvirke analysen er Colcemid ...

Kromosomanalyse er en konventionel teknik udnyttet over hele verden til at diagnosticere kromosom ustabilitet og omrokeringer, der fører til genetiske sygdomme og malignitet 1,2,8,9. Desuden kan en højere opløsning til diagnose og forskning af forfatningsmæssige og kræft-erhvervede genetiske abnormiteter opnås med en kombination af de klassiske cytogenetiske procedurer og molekylære cytogenetiske metoder såsom fluorescens in situ hybridisering (FISH), komparativ genomisk hybridisering (CGH) , og spektral karyotyping (SKY) 14,15. For nylig har disse teknikker blevet brugt til vurdering af kromosom ustabilitet forbundet med stamcelleforskning. Karyotypic abnormiteter såsom aneuploidi af langsigtede dyrkede embryonale celler (ES) og voksne stamceller fra forskellige organismer er blevet rapporteret af flere laboratorier. Nylige undersøgelser understøtter, at nogle cellelinjer er i sagens natur mere tilbøjelige til kromosom instabilitet uanset dyrkningsbetingelser. Af denne grund, når du opretter og / eller opretholde menneske, mus eller Rhesus stamcellelinjer er kromosomanalyse anbefales som en del af kvalitetskontrollen. Mange rapporter beskriver den stigende interesse for brugen af rutine og molekylær cytogenetik at overvåge kromosom stabilitet af stamceller og maligne celler eller forskellige organismer i kultur 10-13. Disse protokoller bliver i stigende grad brugt af nongenetic laboratorier til hurtig kromosomal vurdering af deres dyrkede celler 13. Vi præsenterer vores grundlæggende procedurer for forberedelse kromosom fra forskellige celletyper, som kan anvendes til både kliniske og forskningsmæssige formål og celler afledt af forskellige organismer.

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		<tr height="20">
			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
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chromosome preparation from cultured cells

Kromosom (cytogenetisk) analyse er almindeligt anvendt til påvisning af kromosom ustabilitet. Når efterfulgt af G-banding og molekylære teknikker såsom fluorescens in situ hybridisering (FISH), denne analyse har den magtfulde evne til at analysere de enkelte celler for aberrationer, der involverer gevinst eller tab af dele af genomet og omlejringer involverer et eller flere kromosomer. Hos mennesker forekommer kromosomfejl i cirka 1 per 160 levendefødte 1,2, 60-80% af alle aborter 3,4, 10% af dødfødsler 2,5, 13% af personer med medfødt hjertesygdom 6, 3-6% af infertilitet tilfælde 2, og i mange patienter med forsinkelse udviklingsmæssige og fosterskader 7. Cytogenetisk analyse af malignitet anvendes rutinemæssigt af forskere og klinikere, som observationer af klonede kromosomafvigelser har vist sig at have både diagnostisk og prognostisk betydning 8,9.60, Kromosom isolation er uvurderlig for genterapi og stamcelleforskning af organismer, herunder menneskelige primater og gnavere 10-13. Kromosomer kan isoleres fra celler af levende væv, herunder blod-lymfocytter, hudfibroblaster, amniocyter, placenta, knoglemarv og tumorprøver. Kromosomer analyseres på metafase i mitosen, når de er mest kondenseres og derfor mere tydeligt. Det første trin af kromosomet isolation teknik indebærer afbrydelse af spindel fibre ved inkubation med Colcemid, for at forhindre cellerne i at foretage den efterfølgende anafase fase. Cellerne behandles derefter med en hypotonisk opløsning, og bevaret i kvældet tilstand med Carnoys fiksativ. Cellerne derefter faldt på objektglas og kan derefter anvendes til en række forskellige procedurer. G-banding indebærer trypsinbehandling efterfulgt af farvning med Giemsa at skabe karakteristisk lys og mørke bånd. Det samme procedure at isolere kromosomer kan anvendes til fremstilling af celler til procedurer såsom fluorescens in situ hybridisering (FISH), komparativ genomisk hybridisering (CGH), og spektral karyotyping (SKY) 14,15.

Høj kvalitet metafasespredninger er afgørende for kromosomanalyse. En vellykket analyse giver kromosomer, som er godt spredt, og af passende kromosom morfologi. Korrekt G-banded kromosomer indeholder den karakteristiske lyse og mørke båndmønstre. <img alt="Figur 1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> Figur 1.. En vellykket kromosomal spredning, hvor ...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

Kromosomer kan isoleres fra levende celler, såsom lymfocytter eller hudfibroblaster og fra organismer, herunder mennesker eller mus. Disse kromosom præparater kan yderligere udnyttes til rutinemæssig G-banding og molekylære cytogenetiske procedurer såsom fluorescens in situ hybridisering (FISH), komparativ genomisk hybridisering (CGH), og spektral karyotyping (SKY).

Kromosom Forberedelse Fra dyrkede celler

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

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