Name: slide preparation method to preserve three-dimensional chromatin architecture of testicular germ cells
Uploaded: 2014-01-10T14:15:00
Description: Watch this Scientific Journal Video about Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells at JoVE.com

Jeg takker Jeannie T. Lee for tilsynet med dette projekt, da jeg var i Lee laboratoriet og Tyler Broering for at redigere manuskriptet. Dette arbejde blev støttet af Basil O'Connor Starter Scholar Award fra marts Dimes Foundation og NIH Grant GM098605.

1.3D Forberedelse af dias
<ol><li>Forbered følgende reagenser:<ol>
<li>CSK buffer: 100 mM NaCl, 300 mM saccharose, 10 mM RØR, 3 mM MgCl2. Juster pH til 6,8 med 1 M NaOH. CSK-bufferen opbevares ved 4 °C.</li>
		<li>CSK buffer med 0,5% Triton X-100: Tilsæt 200 μl Triton X-100 til 40 ml CSK buffer. Opløsningen blandes med magnetisk omrører, indtil Triton X-100 er helt opløst. CSK-bufferen opbevares med 0,5% Triton X-100 ved 4 °C.</li>
		<li>4% Paraformaldehyd (PFA)-PBS, pH 7,4: 4 g PFA opløses i ca. 7...

<ol>
	<li>Peters, A. H., Plug, A. W., van Vugt, M. J., de Boer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drying-down+technique+for+the+spreading+of+mammalian+meiocytes+from+the+male+and+female+germline.">A drying-down technique for the spreading of mammalian meiocytes from the male and female germline.</a> Chromosome Res. 5, 66-68 (1997).</li><li>Namekawa, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr Biol. 16, 660-667 (2006).</li><li>Namekawa, S. H., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+nascent+RNA,+single-copy+DNA+and+protein+localization+by+immunoFISH+in+mouse+germ+cells+and+preimplantation+embryos.">Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.</a> Nat Protoc. 6, 270-284 (2011).</li><li>Mahadevaiah, S. K., Costa, Y., Turner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+RNA+FISH+to+study+gene+expression+during+mammalian+meiosis.">Using RNA FISH to study gene expression during mammalian meiosis.</a> Methods in molecular biology. 558, 433-444 (2009).</li><li>Hall, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ectopic+human+XIST+gene+can+induce+chromosome+inactivation+in+postdifferentiation+human+HT-1080+cells.">An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells.</a> Proc Natl Acad Sci U S A. 99, 8677-8682 (2002).</li><li>Huynh, K. D., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+a+pre-inactivated+paternal+X+chromosome+in+early+mouse+embryos.">Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos.</a> Nature. 426, 857-862 (2003).</li><li>Namekawa, S. H., VandeBerg, J. L., McCarrey, J. R., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+chromosome+silencing+in+the+marsupial+male+germ+line.">Sex chromosome silencing in the marsupial male germ line.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9730-9735 (2007).</li><li>Namekawa, S. H., Payer, B., Huynh, K. D., Jaenisch, R., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+imprinted+X+inactivation:+repeat+versus+genic+silencing+in+the+mouse.">Two-step imprinted X inactivation: repeat versus genic silencing in the mouse.</a> Mol Cell Biol. 30, 3187-3205 (2010).</li><li>Ichijima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDC1+directs+chromosome-wide+silencing+of+the+sex+chromosomes+in+male+germ+cells.">MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.</a> Genes Dev. 25, 959-971 (2011).</li><li>Sin, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF8+regulates+active+epigenetic+modifications+and+escape+gene+activation+from+inactive+sex+chromosomes+in+post-meiotic+spermatids.">RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids.</a> Genes Dev. 26, 2737-2748 (2012).</li></ol>

I 3D-diasforberedelse går gennemstrængningstrinnet forud for fikseringstrinnet. Disse trin udføres direkte på seminiferous tubules og derved bevarer den tredimensionelle chromatinstruktur. En alternativ mulighed for at bevare chromatin struktur for RNA / DNA FISH er at udføre permeabilization trin og fiksering trin samtidigt. Denne alternative teknik er blevet udført i pungdyr kimceller og i mus præimplantation embryoner7,8. Men hvis fikseringstrinnet går forud for gennemtrængningstrinnet, kan det giv...

I testikler differentierer kimceller fra diploid spermatogonia gennem meiose til moden, haploid spermatozoa. Under denne proces omformes den nukleare chromatinstruktur af kimceller kontinuerligt og dynamisk. Overfladespredninger er almindeligt anvendt til cytologisk undersøgelse af individuelle spermatogene celler. En fremherskende metode til overfladespredning anvender hypotonisk behandling, hvorved individuelle spermatogene celler spredes og flades1. Disse betingelser er optimale til detaljeret analyse af meiotiske kromosomer. Kromosomale træk såsom synaptisk status og rekombination foci observeres let ved hjælp af denne metode. Den hypotoniske behandling forstyrrer imidlertid subnuklear kromatinarkitektur, således at denne teknik ikke er egnet til strukturel analyse af kerner. Derfor er en forbedret metode designet til at bevare den tredimensionelle chromatinstruktur af testikelkimceller. Denne metode er blevet kaldt den tredimensionale (3D) diasmetode (Figur 1). 3D-diasmetoden har gjort det muligt at detektere spirende RNA-lokalisering i kerner, fordi den oprindeligt blev optimeret til at undersøge genekspression og kromatinestater under testikelkimcelledifferentiering ved RNA-fluorescens in situ-hybridisering (FISH)2,3. Denne 3D-metode gælder også for kombinationen af immunfluorescens, DNA og RNA FISH. Derudover har denne metode hjulpet med opdagelsen af postmeiotisk sexkromati (PMSC), et stille rum af kønskromosomerne, der findes i postmeiotiske spermatids2.
3D-diasmetoden blev oprindeligt optimeret gennem kombinationen af to væsentlige trin i diaspræparatet, der almindeligvis anvendes til nuklear farvning: fikseringstrinet designet til at fastsætte nukleare materialer og gennemtrængningstrinnet, der har til formål at fjerne cytoplasmiske materialer for at forbedre tilgængeligheden af farvningsreagenser, såsom antistoffer og FISH-sonder. Som tidligere beskrevet i en andenpublikation 3er det blevet fastslået, at gennemtrængningstrinnet skal gå forud for fikseringstrinnet for at opnå optimale resultater med lav cytoplasmisk baggrund. I denne 3D-diasmetode udføres gennemtrængningstrinnet og det efterfølgende fikseringstrin direkte på seminiferøse tubules og efterfølges af mekanisk dissociation af kimceller med pincet, før de cytospines på dias. En alternativ metode til RNA FISH af spermatogene celler blev udviklet i et andet laboratorium4. I denne metode, der er i overensstemmelse med 3D-metoden, skal gennemtrængningstrinnet gå forud for fikseringstrinnet for at opnå optimale resultater af RNA FISH.
Følgende protokol beskriver 3D-diasmetoden og giver et eksempel på en mulig applikation, Cot-1 RNA FISH til påvisning af nukleare spirende RNA'er. Cot-1 DNA består af gentagne elementer i genomet. Her hybridiseres en Cot-1 DNA-sonde til en intron og UTR af de spirende udskrifter og registrerer derved de transskriptionsmæssigt aktive regioner i kerne5,6. 3D-dias er alsidige og kan anvendes på en kombination af immunfluorescens, DNA og RNA FISH teknikker med henblik på at opnå en detaljeret undersøgelse af rumlige relationer mellem chromatin arkitektur.

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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cot-1 DNA&#160;</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stratagene Prime-It Fluor Fluorescence Labeling Kit</td>
			<td style="width:147px;">Agilent</td>
			<td align="right" style="width:119px;">
			300380
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Herring sperm DNA Solution</td>
			<td style="width:147px;">Invitrogen</td>
			<td>15634-017</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol, 200 proof (absolute)</td>
			<td>Pharmco-AAPER</td>
			<td align="right">111000200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S2889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Formamide deionized</td>
			<td>American Bioanalytical</td>
			<td>AB00600-00500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate tribasic dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C8532</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bovine serum albumin</td>
			<td style="width:147px;">Lifeblood Medical</td>
			<td style="width:119px;">77110-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran sulfate sodium salt from Leuconostoc spp.</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D6001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">A10491-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P6757</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">M0250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">76240</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">221465</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P4417</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Mounting Medium with DAPI</td>
			<td style="width:147px;">Vector Laboratories</td>
			<td style="width:119px;">H1200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonucleoside-vanadyl complex</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">S1402S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illustra MicroSpin G-50 Columns</td>
			<td>GE Healthcare</td>
			<td style="width:119px;">27-5330-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge</td>
			<td style="width:147px;">Eppendorf</td>
			<td align="right" style="width:119px;">5424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Forceps</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">300-050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge tubes</td>
			<td style="width:147px;">Bioexpress</td>
			<td style="width:119px;">C-3262-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cytospin 3</td>
			<td style="width:147px;">Shandon</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Coplin jar</td>
			<td style="width:147px;">Fisher</td>
			<td style="width:119px;">08-815</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost /Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well dish</td>
			<td>Fisher</td>
			<td style="width:119px;">12-566-301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass&#160;</td>
			<td>Fisher</td>
			<td style="width:119px;">12-544-G</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Air incubator</td>
			<td style="width:147px;">Revolutionary Science</td>
			<td style="width:119px;">RS-IF-203</td>
		</tr>
	</tbody>
</table>

slide preparation method to preserve three-dimensional chromatin architecture of testicular germ cells

Under testikelkimcelledifferentiering ændres strukturen af nuklear kromatin dynamisk. I det følgende beskrives en metode, der er udformet med henblik på at bevare det tredimensionale chromatinarrangement af testikelkimceller, der findes hos mus; Denne metode er blevet kaldt den tredimensionale (3D) diasmetode. I denne metode behandles testikelrør direkte med et gennemtrængningstrin, der fjerner cytoplasmisk materiale, efterfulgt af et fikseringstrin, der løser nukleare materialer. Tubules adskilles derefter, celleaffjedringen er cytospun, og celler klæber til dias. Denne metode forbedrer følsomheden over for påvisning af subnukleare strukturer og gælder for immunfluorescens, DNA og RNA-fluorescens in situ-hybridisering (FISH) og kombinationen af disse detektionsmetoder. Som et eksempel på en mulig anvendelse af 3D-diasmetoden vises en Cot-1 RNA FISH til at registrere spirende RNA'er. 3D-slidemetoden vil lette den detaljerede undersøgelse af rumlige relationer mellem kromatinstruktur, DNA og RNA under testikelkimcelledifferentiering.

Et repræsentativt resultat af et 3D-dias vises i figur 2. I dette eksperiment blev Cot-1 RNA FISH udført for at detektere spirende transskription sammen med immunostaining ved hjælp af anti CBX1 og γH2AX antistoffer. For at kombinere RNA FISH og immunostaining blev immunostaining udført først, efterfulgt af RNA FISH som beskrevet i3. CBX1 er et heterochromatinprotein, der lokaliserer sig til pericentromerisk heterochromatin og tavse kønskromosomer i meiose kaldet en XY-krop (eller sexkr...

Watch this Scientific Journal Video about Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells at JoVE.com

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Slide Forberedelse Metode til at bevare tre-dimensionelle Chromatin Arkitektur testikelceller

Materialet her beskriver en metode udviklet til at bevare den tredimensionelle chromatin struktur testikel kimceller. Dette er blevet kaldt den tredimensionale (3D) diasmetode. Denne metode forbedrer følsomheden for påvisning af subnukleare strukturer og gælder for immunfluorescens, DNA og RNA-fluorescens in situ-hybridisering (FISH).

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

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