Name: chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii
Uploaded: 2018-02-26T13:00:00
Description: Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

Dette arbejde blev støttet af NIGMS (R01GM11755) til P.W.J og af en uddannelse grant stipendium fra National Cancer Institute (NIH) (CA009110) til Niels og J.H.

Alle metoder beskrevet her er blevet godkendt af institutionelle Animal Care og brug udvalg (IACUC) af Johns Hopkins University. Forsøgene blev udført på wild-type C57BL/6J mus.
1. høst føtal eller Neonatal æggestokke og forberedelse af Prophase kromatin spreder
<ol><li>For at udtrække embryoner på 14,5-19,5 dage ofre post-coitum (dpc) de drægtige hundyr via cervikal dislokation eller CO2 kvælning ifølge IACUC retningslinjer. Bemærk: For postnatal dag 1 – 5 ægges...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+all+germ+cells+are+created+equal:+aspects+of+sexual+dimorphism+in+mammalian+meiosis.">Not all germ cells are created equal: aspects of sexual dimorphism in mammalian meiosis.</a> Reproduction. 130 (6), 761-781 (2005).</li><li>Keeney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spo11+and+the+Formation+of+DNA+Double-Strand+Breaks+in+Meiosis.">Spo11 and the Formation of DNA Double-Strand Breaks in Meiosis.</a> Genome Dyn Stab. 2, 81-123 (2008).</li><li>Zickler, D., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination,+Pairing,+and+Synapsis+of+Homologs+during+Meiosis.">Recombination, Pairing, and Synapsis of Homologs during Meiosis.</a> Cold Spring Harb Perspect Biol. 7 (6), (2015).</li><li>Vries, F. A., 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=Mouse+Sycp1+functions+in+synaptonemal+complex+assembly,+meiotic+recombination,+and+XY+body+formation.">Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation.</a> Genes Dev. 19 (11), 1376-1389 (2005).</li><li>Baker, S. M., 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=Involvement+of+mouse+Mlh1+in+DNA+mismatch+repair+and+meiotic+crossing+over.">Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over.</a> Nat Genet. 13 (3), 336-342 (1996).</li><li>Lipkin, S. M., 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=Meiotic+arrest+and+aneuploidy+in+MLH3-deficient+mice.">Meiotic arrest and aneuploidy in MLH3-deficient mice.</a> Nat Genet. 31 (4), 385-390 (2002).</li><li>Kolas, N. K., 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=Localization+of+MMR+proteins+on+meiotic+chromosomes+in+mice+indicates+distinct+functions+during+prophase+I.">Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I.</a> J Cell Biol. 171 (3), 447-458 (2005).</li><li>Rankin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+elaboration:+making+sense+of+meiotic+cohesin+dynamics.">Complex elaboration: making sense of meiotic cohesin dynamics.</a> FEBS Journal. 282 (13), 2426-2443 (2015).</li><li>Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Cohesin+and+Condensin+in+Chromosome+Dynamics+During+Mammalian+Meiosis.">Roles of Cohesin and Condensin in Chromosome Dynamics During Mammalian Meiosis.</a> J. Reprod Dev. 59 (5), 431-436 (2013).</li><li>Verver, D. E., Hwang, G. H., Jordan, P. W., Hamer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+complex+chromosome+structures+during+meiosis:+versatile+deployment+of+Smc5/6.">Resolving complex chromosome structures during meiosis: versatile deployment of Smc5/6.</a> Chromosoma. 125 (1), 15-27 (2016).</li><li>Hopkins, J., 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=Meiosis-specific+cohesin+component,+Stag3+is+essential+for+maintaining+centromere+chromatid+cohesion,+and+required+for+DNA+repair+and+synapsis+between+homologous+chromosomes.">Meiosis-specific cohesin component, Stag3 is essential for maintaining centromere chromatid cohesion, and required for DNA repair and synapsis between homologous chromosomes.</a> PLoS Genet. 10 (7), e1004413 (2014).</li><li>Hwang, G., 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=SMC5/6+is+required+for+the+formation+of+segregation-competent+bivalent+chromosomes+during+meiosis+I+in+mouse+oocytes.">SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.</a> Development. 144 (9), 1648-1660 (2017).</li><li>Kota, S. K., Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+transitions+in+germ+cell+development+and+meiosis.">Epigenetic transitions in germ cell development and meiosis.</a> Dev Cell. 19 (5), 675-686 (2010).</li><li>Taketo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microspread+ovarian+cell+preparations+for+the+analysis+of+meiotic+prophase+progression+in+oocytes+with+improved+recovery+by+cytospin+centrifugation.">Microspread ovarian cell preparations for the analysis of meiotic prophase progression in oocytes with improved recovery by cytospin centrifugation.</a> Methods Mol Biol. 825 (1), 173-181 (2012).</li><li>Sun, X., Cohen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+recombination+in+mouse+oocytes.">Studying recombination in mouse oocytes.</a> Methods Mol Biol. 957 (1), 1-18 (2013).</li><li>Kim, J. H., Ishiguro, K., Kudo, N., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+meiosis-specific+cohesins+in+mouse+embryonic+oocytes.">Studying meiosis-specific cohesins in mouse embryonic oocytes.</a> Methods Mol Biol. 957 (1), 47-57 (2013).</li><li>Susiarjo, M., Rubio, C., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+mammalian+female+meiosis.">Analyzing mammalian female meiosis.</a> Methods Mol Biol. 558, 339-354 (2009).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nat Rev Genet. 2 (4), 280-291 (2001).</li><li>Hassold, T., Hall, H., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+human+aneuploidy:+where+we+have+been,+where+we+are+going.">The origin of human aneuploidy: where we have been, where we are going.</a> Hum Mol Genet. 16, R203-R208 (2007).</li><li>Pincus, G., Enzmann, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Comparative+Behavior+of+Mammalian+Eggs+In+vivo+and+In+vitro:+I.+The+Activation+of+Ovarian+Eggs.">The Comparative Behavior of Mammalian Eggs In vivo and In vitro: I. The Activation of Ovarian Eggs.</a> J Exp Med. 62 (5), 665-675 (1935).</li><li>Chambon, J. P., Hached, K., Wassmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+spreads+with+centromere+staining+in+mouse+oocytes.">Chromosome spreads with centromere staining in mouse oocytes.</a> Methods Mol Biol. 957 (1), 203-212 (2013).</li><li>Stein, P., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+oocyte+microinjection,+maturation+and+ploidy+assessment.">Mouse oocyte microinjection, maturation and ploidy assessment.</a> J Vis Exp. (53), (2011).</li></ol>

Kromatin spredning præparater giver forskere at kronologisk studere kvindelige pattedyr meiose og den dynamiske lokalisering af proteiner involveret. Embryonale og neonatal kromatin opslag giver mulighed for tæt analyse af hændelser i hele meiotiske prophase. Metafase jeg og metafase II kromatin spreads kan bruges til at skelne enkelt Søster kromatider fra parret søstre og parret homologe kromosomer, samt vurdere Ploidi. Til sammenligning, kan den protokol, der er beskrevet her være fordelagtigt i forhold til hele ...

Under spermatogenesen, er store semisynkron bølger af meiotiske kønsceller hurtigt og kontinuerligt genopfyldes i testiklerne i starten af puberteten og hele voksenlivet1. I modsætning til mænd initieres meiose hos kvinder alene i løbet af fostrets udvikling. Efter fødslen, oocyter fortsat arresteret i en langvarig dictyate fase af prophase jeg med en intakt germinale vesikel (GV; nukleare kuvert) indtil puberteten. Ved begyndelsen af puberteten vælges en delmængde af oocytter cyklisk underkastes vækst og modning, markerer indledningen af meiotiske genoptagelse. Meiotiske genoptagelse i fuldt udvokset oocyter er manifesteret ved forsvinden af GV i en proces kendt som germinale vesikel opdeling (GVBD). Oocyt derefter gennemgår kromosom kondens og segregation, efterfulgt af polar body ekstrudering. Oocytter blive pågrebet progression til MII og er stimuleret til at fuldføre den anden og sidste meiotiske deling kun efter befrugtningen.
Kvindelige frugtbarhed er stærkt afhængige af succesen af meiotiske prophase jeg progression. Nøglen til dette er dannelsen af en fysisk sammenhæng mellem homologe kromosomer kendt som chiasmata, hvilket er medieret af reparation af inducerede DNA dobbeltstrenget pauser (DSBs) via crossover rekombination2. Denne proces sker i forbindelse med en dynamisk protein-rige stillads kendt som synaptonemal complex (SC), der danner mellem homologe kromosomer at lette deres synapsis3. SC er en lynlås-lignende tredelte struktur, der består af to parallelle laterale elementer forbundet af centrale region proteiner der holder homologs sammen i hele igangværende DNA reparation. Før synapsis danner prækursorer af de laterale elementer, kaldet aksial elementer, mellem Søster kromatider. Synaptonemal komplekse proteiner som SYCP2 og SYCP3 udgør aksial elementer, der colocalize til søster-chromatid samhørighed akser under tidlige prophase. Disse senere fungere som bindende websteder for tværgående filament protein, SYCP1, der letter centrale element forsamling og synapsis mellem justeret homologs4. I mus oocytter, er komplet synapsis indiceret ved tilstedeværelsen af 20 helt overlappende SYCP3 og SYCP1 strækninger, som kan visualiseres ved hjælp af kromatin sprede præparater. Synapsis er afsluttet træder i pachytene underniveau, hvorved modne delefiltre, der er bestemt til form chiasmata mellem homologs er dekoreret med mutL homolog (MLH1/3) dimerer at fremme deres korrekte behandling5,6 , 7. den strukturelle vedligeholdelse af kromosomer (SMC) komplekser, herunder cohesin, condensin og SMC5/6 komplekse, er vigtig for reguleringen af kromosom dynamik og struktur i hele meiose8,9, 10,11,12. Kollektivt, sikre disse begivenheder ordentlig bi-orientering af homologe kromosomer til modsatrettede spindel polakker efter afmontering af Overvågningsudvalgets
Meiotiske cellecyklus er en kraftfuld model til at undersøge forskellige proteiner i genom vedligeholdelse på grund af den programmerede induktion og efterfølgende reparation af DNA DSBs roller. Derudover er pattedyr meiose også en relevant model for studiet af epigenetiske ændringer og prægning13. Men, det er teknisk vanskeligt at vurdere disse begivenheder under kvindelige meiose, der foregår i føtal og neonatal æggestokkene hos pattedyr (figur 1). Prophase jeg kan opdeles i 5 substages: leptonema, zygonema, pachynema, diplonema og dictyate. Heri, beskriver vi hvordan man isolere og skelne mellem fostrets og neonatal æggestokke og testikler (figur 2). Tilpasset fra tidligere beskrevne metoder, dette manuskript også skitserer en protokol (trin 1) med video demonstration for forberedelse af kvindelige meiotiske prophase jeg kromatin spreder14,15,16,17 . Når kombineret med immunolabelling, som beskrevet i trin 6-7, denne protokol giver mulighed for detaljeret mikroskopisk analyse af prophase jeg begivenheder i oocytter.
Oogenesen er fejlbehæftet, og kromosom missegregation begivenheder i løbet af den første meiotiske deling repræsenterer den mest almindelige kilde til genetisk sygdom i afkom18,19. I dette manuskript (protokol trin 2) beskriver vi en protokol, hvor modne GV-iscenesatte oocyter er udvundet fra PRIMES æggestokkene af voksne hunmus. Betingelser, støttende gennemgå fuldt udvokset oocyter luteiniserende hormon-uafhængige genoptagelse af meiose efter isolation og kultur20. Efter meiotiske genoptagelse, oocyter fremskridt gennem meiose, derefter anholde på metafase II. Oocytter fortsat arresteret på metafase II, medmindre befrugtet. I trin 2 – 5, tilpasser vi tidligere rapporteret protokoller med video demonstration at beskrive, hvordan at indsamle, kultur og forberede kromatin sprede præparater21MI og MII oocyter. Denne kromatin spredning teknik giver mulighed for klare immunolabelling af proteiner tilknyttet kromosomer. Desuden, denne protokol kan også bruges til at skelne mellem bivalent og univalent kromosomer, og kan yderligere løse enkelt Søster kromatider at lette vurderingen af oocyt Ploidi. Derfor, ud over afslørende lokalisering mønstre af meiotiske proteiner, denne protokol kan også tjene som et uvurderligt værktøj for belyse mulige årsager til kromosom missegregation under MI og MII.

<table>
	<tbody>
		<tr>
			<td>10X Phosphate buffered saline (PBS) pH 7.4</td>
			<td>Quality Biological</td>
			<td>119-069-161</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm x 15 mm petri dishes</td>
			<td>Denville</td>
			<td>T1106</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm x 12mm petri dishes</td>
			<td>Denville</td>
			<td>T1103</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>VWR</td>
			<td>300-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissection scissors</td>
			<td>Ted Pella</td>
			<td>1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160;scissors, sharp tip, 41/2&#34;</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass square 1 5/8</td>
			<td>Carolina Biological Supply Company</td>
			<td>742300</td>
			<td>Autoclave before each use</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S8501</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium Citrate Dihydrate</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>646563</td>
			<td>Add to hypotonic buffer immediately before use</td>
		</tr>
		<tr>
			<td>50x Protease Inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Add to hypotonic buffer immediately before use</td>
		</tr>
		<tr>
			<td>Turberculin syringe with needle (1cc, 27 G x 1/2 in)</td>
			<td>Becton, Dickinson (BD)</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold seal ultra frost glass slides (25X75mm, 1mm thick)</td>
			<td>Thermo</td>
			<td>3063-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP pen, large (liquid blocker)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>71310</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde aqueous</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Detergent in manuscript</td>
		</tr>
		<tr>
			<td>Immuno stain moisture chamber</td>
			<td>Evergreen</td>
			<td>240-9020-Z10</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jars</td>
			<td>Fisher</td>
			<td>08-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-flo 200</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>74257</td>
			<td>Wetting agent in manuscript</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin (PMSG)</td>
			<td>Sigma</td>
			<td>G4877</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin (HCG)</td>
			<td>Sigma</td>
			<td>C0434</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>PYREX Spot Plates with nine concave cavities</td>
			<td>Fisher</td>
			<td>13-748B</td>
			<td>Autoclave before each use</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>Fisher</td>
			<td>22260943</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Brand Parafilm M</td>
			<td>Sigma</td>
			<td>BR701501</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Latex rubber tubing (3.2 mm inner diameter x 6.4 mm outer diameter)</td>
			<td>Fisher</td>
			<td>14-178-5B</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Latex rubber tubing (6.4 mm inner diameter x 11.1 mm outer diameter)</td>
			<td>Fisher</td>
			<td>14-178-5D</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Flexible silicone rubber nosepiece, hard plastic mouthpiece and 15 inches of latex tubing</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Mitutoyo micrometer head</td>
			<td>Mituoyo</td>
			<td>150-208</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BD Biosciences</td>
			<td>309646</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Syringe filter 0.45 &#956;m filter</td>
			<td>Corning</td>
			<td>431220</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipette 5 3/4&#34;</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>83 x 0.5 mm pipette tip</td>
			<td>Denville</td>
			<td>P3080</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Invitrogen</td>
			<td>11415049</td>
			<td></td>
		</tr>
		<tr>
			<td>Waymouth&#39;s media</td>
			<td>Life Technologies</td>
			<td>11220035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher</td>
			<td>A3160602</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A3311</td>
			<td>For oocyte culture media</td>
		</tr>
		<tr>
			<td>500ml filter units 0.22um</td>
			<td>Denville</td>
			<td>F5227</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode&#8217;s solution</td>
			<td>Sigma</td>
			<td>T1788</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma</td>
			<td>H-1270</td>
			<td>For ADB/wash buffer</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td>For ADB/wash buffer</td>
		</tr>
		<tr>
			<td>VECTASHIELD antifade mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides (22mmx60mm)</td>
			<td>Fisher</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SteREO Discovery.V8</td>
			<td>Zeiss</td>
			<td>495015-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Observer Z1</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-MLH1</td>
			<td>Life Technologies</td>
			<td>MA5-15431</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti-SYCP1</td>
			<td>Life Technologies</td>
			<td>PA1-16763</td>
			<td>Dilution factor- 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-SCP3</td>
			<td>Santa Cruz</td>
			<td>sc-20845</td>
			<td>Dilution factor- 1:50</td>
		</tr>
		<tr>
			<td>Human anti-centromere protein</td>
			<td>Antibodies Incorporated</td>
			<td>15-235</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti- TOPOII</td>
			<td>Abcam</td>
			<td>ab109524</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Mouse anti-Histone H4 (di methyl K20, tri methyl K20)</td>
			<td>Abcam</td>
			<td>ab78517</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Rec8</td>
			<td>Courtesy of Dr. Karen Schindler</td>
			<td>N/A</td>
			<td>Dilution factor- 1:10,000</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11057</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-21202</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-31573</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11004</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11013</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11011</td>
			<td>Dilution factor- 1:500</td>
		</tr>
	</tbody>
</table>

chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii

Kromatin udbredelsen teknikker har været meget anvendt til at vurdere den dynamiske lokalisering af forskellige proteiner under gametogenesis, især for spermatogenesen. Disse teknikker giver mulighed for visualisering af proteiner og DNA lokalisering mønstre under meiotiske begivenheder såsom homologe kromosom parring, synapsis og DNA reparation. Mens nogle protokoller er blevet beskrevet i litteraturen, er generelle kromatin udbredelsen teknikker ved hjælp af pattedyr prophase oocyter begrænset og vanskelig på grund af timingen af meiose indledning i føtal æggestokke. I sammenligning, kan prophase spermatocytes afhentes fra unge mandlige mus med højere udbytter uden behov for microdissection. Det er imidlertid vanskeligt at opnå en ren synkroniserede befolkning af celler på bestemte stadier på grund af heterogenitet af meiotiske og post meiotiske kønscelle befolkninger i de unge og voksne testis. I senere faser af meiose er det fordelagtigt at vurdere oocyter gennemgår meiose jeg (MI) eller meiose II (MII), fordi grupper af modne oocyter kan indsamles fra voksne hunmus og stimuleres til at genoptage meiose i kultur. Her, metoder til meiotiske kromatin sprede præparater ved hjælp af oocytter dissekeret fra fostrets, neonatal og voksne æggestokkene er beskrevet med ledsagende video demonstrationer. Kromosom missegregation begivenheder i pattedyr oocyter er hyppige, især under MI. Disse teknikker kan bruges til at vurdere og karakterisere effekten af forskellige mutationer eller miljømæssige eksponeringer under forskellige faser af oogenesen. Som der er tydelige forskelle mellem oogenesen og spermatogenesen, er de teknikker, der er beskrevet inden for uvurderligt for at øge vores forståelse af pattedyr oogenesen og kromosom og protein dynamics seksuelt dimorfe funktioner under meiose .

Vi har beskrevet to teknikker til visualisering og vurdere meiotiske kromosomer i oocytter. Den første teknik er dækket mod vurdere prophase progression i embryonale og neonatal æggestokke. Prophase kromatin spredning præparater er utrolig værdifuld til at visualisere mange dynamiske processer under meiose, herunder kromosom parring, synapsis og desynapsis, homologe rekombination, og epigenetiske kromosom remodellering. Her, vi har vist nytten af denne metode til robust visualisering...

Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Kromatin sprede præparater til analyse af musen oocyt Progression fra Prophase til metafase II

Oogenesen i pattedyr er kendt for at være fejlbehæftet, især på grund af kromosom missegregation. Dette manuskript beskriver kromatin spredes forberedelse metoder til mus prophase, metafase I og II-iscenesatte oocyter. Disse grundlæggende teknikker giver mulighed for undersøgelse af kromatin-bundet proteiner og kromosom morfologi i hele pattedyr oogenesen.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

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Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Sekventiel Salt ekstraktioner til analyse af Bulk kromatin bindende egenskaber af kromatin ændre komplekser

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Analyse af c-KIT ligand-promotoren ved anvendelse af chromatinimmunpræcipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

Chromatin-immunpræcipitation (ChIP) i mus-T-cellelinier

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Retroviral Scanning: Kortlægning af MLV Integration Sites til at definere Cell-specifikke Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

Generation af indfødte kromatin Immunoprecipitation sekventering biblioteker for Nucleosome tæthed analyse

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Konstituerende og inducerbar systemer for genetiske In Vivo ændring af mus hepatocytter brug af hydrodynamiske hale vene injektion

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Generation af Genome-wide kromatin kropsbygning Capture biblioteker fra stramt iscenesatte tidlige Drosophila embryoner

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Kromatin Immunoprecipitation (ChIP) af Histon ændringer fra Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

Tilpasning af hybridisering fange af kromatin-associerede proteiner for Proteomics til pattedyrceller

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Formaldehyd-assisteret Isolation af regulerende elementer til foranstaltning kromatin tilgængelighed i pattedyrceller

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...