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.

Dit protocol werd goedgekeurd door de Institutional Animal Care en gebruik Comite (IACUC) aan de Universiteit van Georgia. 1. Voorbereidingen <ol><li> Voor eicel cultuur aankoop of vers bereiden Minimaal Essentieel Medium (MEM) en aangevuld met 3 mg / ml runderserumalbumine (BSA) als aangegeven in tabel 1. Plaats een polystyreen fles op een laad- saldo (tarra nul). Voeg alle reagentia, behalve BSA, teneinde brengen en het uiteindelijke volume met MQ-water ...

<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>

Hoewel er meerdere werkwijzen voor het exogene nucleïnezuur overdracht somatische cellen, zoals elektroporatie en transfectie, microinjectie is de optimale methode voor afgifte van RNA moleculen in transcriptioneel rustende muizen oöcyten. Het huidige protocol levert een effectieve aanpak voor in vitro depletie van specifieke mRNAs die de functionele testen van verschillende spil en / of MTOC geassocieerde factoren bij oöcyten mogelijk. Deze aanpak resulteert in een efficiënte transcript uitputting en is ze...

Meiose is een unieke divisie proces dat plaatsvindt in gameten (eicellen en zaadcellen) en omvat twee opeenvolgende divisies zonder tussenliggende DNA-synthese van homologe chromosomen en zusterchromatiden scheiden tijdens de meiose I en meiose II, respectievelijk 1. Fouten in chromosoom segregatie tijdens de meiose verdeeldheid in eicellen kan leiden tot aneuploïdie, die wordt overgenomen door het embryo tijdens de bevruchting. Met name de incidentie van aneuploïdie in ontwikkelende embryo&#39;s stijgt met voortschrijdende leeftijd van de moeder en is een hoofdoorzaak van aangeboren afwijkingen en miskramen 1,2, dus het benadrukken een grote behoefte om de moleculaire basis van aneuploïdie in meiotische deling begrijpen . Tijdens de celdeling, chromosoom segregatie is sterk afhankelijk van de montage van de microtubule spoelfiguur en de vestiging van stabiele chromosoom-microtubuli interacties voor de juiste bevestiging aan tegenovergestelde spindelpalen. Belangrijker nog, meiotische spindel formatie in zoogdieren eicellen verschilt van mitose in somatische cellen, en wordt gereguleerd door unieke microtubule organiserende centra (MTOCs) dat centriolen 3,4 missen. Essentiële eiwitten die nodig zijn voor microtubuli nucleatie en organisatie lokaliseren aan eicel MTOCs, waaronder γ-tubuline dat microtubule assemblage katalyseert. Bovendien pericentrin functioneert als essentiële steigers eiwit, dat bindt en ankers γ-tubuline alsook andere factoren MTOCs 5. Met name onze studies aangetoond dat depletie van belangrijke MTOC-geassocieerde eiwitten verstoort meiotische spindel organisatie en leidt tot chromosoomsegregatie fouten eicellen, die niet volledig opgelost door de spileenheid checkpoint (SAC) 6,7. Daarom defecten in spindel stabiliteit, die niet leiden tot meiotische arrestatie, vormen een groot risico bij te dragen aan aneuploïdie. Ondanks hun essentiële rol in de assemblage van de spoel en organisatie, eicel MTOC protein samenstelling en functie blijft slecht begrepen. Het testen van de functie van de specifieke doelgroep eiwitten in zoogdieren eicellen is een uitdaging, omdat de cellen transcriptie rusttoestand kort voor de hervatting van de meiose 8,9. Vandaar pre-ovulatoire eicellen afhankelijk maternale mRNA winkels meiose hervatten en te ondersteunen meiotische deling en de eerste splitsing divisies na bevruchting 10,11. De werkzaamheid van RNA-interferentie (RNAi) gemedieerde afbraak van mRNA transcripten in zoogdieren eicellen is goed ingeburgerd en moedersterfte RNA&#39;s geworven voor de vertaling tijdens de meiose rijping zijn bijzonder vatbaar voor siRNA gericht 12-14. Daarom, micro-injectie van korte interfererende RNA&#39;s (siRNA&#39;s) in de eicellen levert een waardevolle aanpak doel mRNA afbrekende voor functionele testen. We beschrijven hier werkwijzen voor de isolatie van muizen eicellen en siRNA-gemedieerde depletie specifieke transcripts de functie van wezenlijke MTOC-geassocieerde eiwit te testen, pericentrin. Verder beschrijven we immunofluorescentieanalyse voorwaarden meiotische spindel formatie in oöcyten evalueren.

<table border="0">
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		<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.

Micro-injectie van siRNAs een effectieve aanpak voor mRNA afbraak en daaropvolgende eiwit depletie in oöcyten die efficiënt en zeer specifieke functionele testen van verschillende doelwit factoren in vitro mogelijk maakt. Vervolgens wordt immunofluorescentie gebruikt voor specifieke fenotype analyse en eiwit uitputting valideren siRNA-geïnjecteerde oöcyten. In het huidige voorbeeld, fluorescent etikettering van individuele eicellen met DAPI samen met anti-tubuline en anti-pericentrin antilichamen ingeschake...

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

Hier presenteren we een protocol specifieke siRNA-gemedieerde mRNA afbraak gevolgd door immunofluorescentie analyse meiotische spileenheid en organisatie evalueren muizen oöcyten. Dit protocol is geschikt voor in vitro depletie van transcripties en functionele beoordeling van verschillende spil en / of MTOC geassocieerde factoren in oöcyten.

Meiotische Spil Assessment in Muis Eicellen door siRNA-gemedieerde 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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