Name: meiotic spindle assessment in mouse oocytes by sirna-mediated silencing
Uploaded: 2015-10-11T14:00:00
Description: Watch this Scientific Journal Video about Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing at JoVE.com

This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.

Denne protokol blev godkendt af Institutional Animal Care og brug Udvalg (IACUC) ved University of Georgia. 1. Forberedelser <ol><li> For oocyt kultur, køb eller forberede frisk Minimal Essential Medium (MEM) og supplerer med 3 mg / ml bovint serumalbumin (BSA) som vist i tabel 1. Placer en polystyren flaske på en loading balance (tara til nul). Tilføj alle reagenser, undtagen BSA, i orden og opdrage det endelige volumen med MQ-vand efter vægt til i alt 250 g. Tilsæt ...

<ol>
	<li>Nagaoka, S. I., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aneuploidy:+mechanisms+and+new+insights+into+an+age-old+problem.">Human aneuploidy: mechanisms and new insights into an age-old problem.</a> Nat Rev Genet. 13 (7), 493-504 (2012).</li><li>Hassold, T. J., Hunt, P. A. <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>Szollosi, D., Calarco, P., Donahue, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+Centrioles+in+the+First+and+Second+Meiotic+Spindles+of+Mouse+Oocytes.">Absence of Centrioles in the First and Second Meiotic Spindles of Mouse Oocytes.</a> J Cell Sci. 11 (2), 521-541 (1972).</li><li>Manandhar, G., Schatten, H., Sutovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+Reduction+During+Gametogenesis+and+Its+Significance.">Centrosome Reduction During Gametogenesis and Its Significance.</a> Biol Reprod. 72 (1), 2-13 (2005).</li><li>Zimmerman, W. C., Sillibourne, J., Rosa, J., Doxsey, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis-specific+anchoring+of+gamma+tubulin+complexes+by+pericentrin+controls+spindle+organization+and+mitotic+entry.">Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.</a> Mol Biol Cell. 15 (8), 3642-3647 (2004).</li><li>Ma, W., Baumann, C., Viveiros, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NEDD1+is+crucial+for+meiotic+spindle+stabilty+and+accurate+chromosome+segregation+in+mammalian+oocytes.">NEDD1 is crucial for meiotic spindle stabilty and accurate chromosome segregation in mammalian oocytes.</a> Dev Biol. 339 (439-450), (2010).</li><li>Ma, W., Viveiros, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+pericentrin+in+mouse+oocytes+disrupts+microtubule+organizing+center+function+and+meiotic+spindle+organization.">Depletion of pericentrin in mouse oocytes disrupts microtubule organizing center function and meiotic spindle organization.</a> Mol Reprod Dev. 81 (11), 1019-1029 (2014).</li><li>Bouniol-Baly, C., 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=Differential+Transcriptional+Activity+Associated+with+Chromatin+Configuration+in+Fully+Grown+Mouse+Germinal+Vesicle+Oocytes.">Differential Transcriptional Activity Associated with Chromatin Configuration in Fully Grown Mouse Germinal Vesicle Oocytes.</a> Biol Reprod. 60 (3), 580-587 (1999).</li><li>De La Fuente, R., Eppig, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Activity+of+the+Mouse+Oocyte+Genome:+Companion+Granulosa+Cells+Modulate+Transcription+and+Chromatin+Remodeling.">Transcriptional Activity of the Mouse Oocyte Genome: Companion Granulosa Cells Modulate Transcription and Chromatin Remodeling.</a> Dev Biol. 229 (1), 224-236 (2001).</li><li>Hodgman, R., Tay, J., Mendez, R., Richter, 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=CPEB+phosphorylation+and+cytoplasmic+polyadenylation+are+catalyzed+by+the+kinase+IAK1/Eg2+in+maturing+mouse+oocytes.">CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes.</a> Development. 128 (14), 2815-2822 (2001).</li><li>De La Fuente, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+in+the+germinal+vesicle+(GV)+of+mammalian+oocytes.">Chromatin modifications in the germinal vesicle (GV) of mammalian oocytes.</a> Dev Biol. 292 (1), 1-12 (2006).</li><li>Wianny, F., Zernicka-Goetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+interference+with+gene+function+by+double-stranded+RNA+in+early+mouse+development.">Specific interference with gene function by double-stranded RNA in early mouse development.</a> Nat Cell Biol. , 270-275 (2000).</li><li>Svoboda, P., Stein, P., Hayashi, H., Schultz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+reduction+of+dormant+maternal+mRNAs+in+mouse+oocytes+by+RNA+interference.">Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference.</a> Development. 127 (19), 4147-4156 (2000).</li><li>Svoboda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renaissance+of+mammalian+endogenous+RNAi.">Renaissance of mammalian endogenous RNAi.</a> FEBS Letters. 588 (15), 2550-2556 (1016).</li><li>Behringer, R., Gertsenstein, M., Nagy, K., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2014).</li></ol>

Mens der er flere metoder til eksogen nukleinsyre overførsel til somatiske celler, såsom elektroporering og transfektion, mikroinjektion er den optimale metode til levering af RNA-molekyler i transkriptionelt hvilende museoocytter. Den nuværende protokol giver en effektiv metode til in vitro-udtømning af specifikke mRNA, der giver den funktionelle test af forskellige spindel og / eller MTOC-associerede faktorer i oocytter. Denne tilgang resulterer i en effektiv udskrift udtynding og er meget fleksibel. Selv...

Meiose er en unik division proces, der forekommer i gameter (oocytter og sæd) og involverer to på hinanden følgende opdelinger uden mellemliggende DNA-syntese at adskille homologe kromosomer og søsterchromatider under meiose-I og meiose-II, henholdsvis 1. Fejl i kromosom adskillelse under meiotiske deling i oocytter kan resultere i aneuploidi, som er arvet af fosteret under befrugtningen. Især forekomsten af aneuploidi i udviklingslandene embryoner stiger med fremrykkende moderens alder og er en væsentlig årsag til medfødte fødselsdefekter samt abort hos kvinder 1,2, således understreger et stort behov for at forstå den molekylære basis for aneuploidi i meiotiske deling . Under celledeling, kromosom adskillelse er helt afhængig af samlingen af ​​mikrotubuli spindel apparater og etablering af stabile kromosom-mikrotubuli interaktioner for korrekt fastgørelse til modsatte spindel denpoler. Vigtigere, meiotiske spindel dannelse i pattedyr oocytter adskiller sig fra mitosen i somatiske celler, og reguleres af unikke mikrotubulus-organisering centre (MTOCs) der mangler centrioler 3,4. Essentielle proteiner er nødvendige for mikrotubuli nukleering og organisering lokalisere til oocyt MTOCs, herunder γ-tubulin som katalyserer mikrotubulus-samling. Desuden pericentrin fungerer som en væsentlig stillads protein, som binder og ankre y-tubulin samt andre faktorer på MTOCs 5. Især vores undersøgelser viser, at udtømning af vigtige MTOC-associerede proteiner forstyrrer meiotisk spindel organisation og fører til kromosom segregation fejl i oocytter, som ikke er fuldt løst ved spindlen forsamling kontrolpunkt (SAC) 6,7. Derfor mangler i spindel stabilitet, der ikke udløser meiotisk anholdelse, udgør en betydelig risiko i at bidrage til aneuploidi. På trods af deres afgørende rolle i spindel samling og organisation, oocyt MTOC protEin sammensætning og funktion forbliver dårligt forstået. Test af funktion af specifikke target proteiner i mammale oocytter er udfordrende, da cellerne bliver transkriptionelt hvilende kort før genoptagelsen af meiose 8,9. Derfor præovulatoriske oocytter afhængige maternale mRNA butikker for at genoptage meiose og støtte meiotiske deling samt de første spaltningsprodukter divisioner efter befrugtningen 10,11. Effekten af RNA-interferens (RNAi) medieret nedbrydning af mRNA-transkripter i mammale oocytter er veletableret og maternelle RNA&#39;er rekrutteret til oversættelse under meiotisk modning er særligt modtagelige for siRNA rettet mod 12-14. Derfor mikroinjektion af korte interfererende RNA (siRNA&#39;er) i oocyter giver en værdifuld tilgang til at udtømme mål mRNA til funktionel test. Her beskriver vi fremgangsmåder til isolering af museoocytter og siRNA-medieret nedbrydning af specifikke transcriPTS for at teste funktionen af ​​en væsentlig MTOC-associeret protein, pericentrin. Desuden beskriver vi immunofluorescensanalyse betingelser at evaluere meiotisk spindeldannelse i oocytter.

<table border="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pregnant Mare&#39;s Serum Gonadotropin (PMSG)</td>
			<td>EMD Biosciences</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal Essential Medium (MEM)</td>
			<td></td>
			<td></td>
			<td>*Recipe outlined in Table 1</td>
		</tr>
		<tr>
			<td>Earle&#39;s Balanced Salt Solution (10x)</td>
			<td>Sigma</td>
			<td>E-7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S-5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic Acid, sodium salt&#160;</td>
			<td>Sigma</td>
			<td>P-5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G, potassium salt&#160;</td>
			<td>Sigma</td>
			<td>P-7794</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Sigma</td>
			<td>S-9137</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Sigma</td>
			<td>G-8540</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, disodium salt dihydrate&#160;</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential Amino Acids (50x)</td>
			<td>Gibco&#160;</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamin Mixture (100x)</td>
			<td>Sigma</td>
			<td>M-6895</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red solution</td>
			<td>Sigma</td>
			<td>P-0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td></td>
		</tr>
		<tr>
			<td>Milrinone</td>
			<td>Sigma</td>
			<td>M4659</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.01</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax M2 Media with Hepes</td>
			<td>EMD Millipore</td>
			<td>MR-015-D</td>
			<td></td>
		</tr>
		<tr>
			<td>siRNAs targeting Pericentrin</td>
			<td>Qiagen</td>
			<td>GS18541</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative control siRNAs&#160;</td>
			<td>Qiagen</td>
			<td>SI03650318</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Hyclone</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Pericentrin (rabbit)</td>
			<td>Covance</td>
			<td>PRB-432C</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetylated a-tubulin (mouse)</td>
			<td>Sigma</td>
			<td>T-6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-21430</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti -mouse Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Major Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope (SMZ 800)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Fluorescent Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet Micro-injections System</td>
			<td>Eppenforf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro manipulators</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-injection needles (femtotips)</td>
			<td>Eppendorf</td>
			<td>930000035</td>
			<td></td>
		</tr>
		<tr>
			<td>Holding pipettes (VacuTip)</td>
			<td>Eppendorf</td>
			<td>930001015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm culture dishes</td>
			<td>Corning Life Sciences</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3367</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 mm CA Filter System</td>
			<td>Corning Life Sciences</td>
			<td>430768</td>
			<td></td>
		</tr>
	</tbody>
</table>

meiotic spindle assessment in mouse oocytes by sirna-mediated silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Mikroinjektion af siRNA&#39;er giver en effektiv metode til mRNA nedbrydning og efterfølgende protein udtynding i oocytter, der muliggør effektiv og meget specifik funktionel test af forskellige målgrupper faktorer in vitro. Efterfølgende immunofluorescens anvendes til særlige fænotype-analyse samt at validere protein udtynding i siRNA-injicerede oocytter. I den nuværende eksempel fluorescerende mærkning af individuelle oocytter med DAPI sammen med anti-tubulin og anti-pericentrin antistoffer aktiveret:...

Watch this Scientific Journal Video about Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing at JoVE.com

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Her præsenterer vi en protokol for specifik siRNA-medieret mRNA udtømning efterfulgt af immunofluorescensanalyse at evaluere meiotisk spindel samling og organisation i museoocytter. Denne protokol er velegnet til in vitro-udtømning af udskrifter og funktionel vurdering af forskellige spindel og / eller MTOC-associerede faktorer i oocytter.

Meiotiske Spindel Vurdering i museoocytter af siRNA-medieret Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

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