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 protokollen ble godkjent av Institutional Animal Care og bruk Committee (IACUC) ved University of Georgia. 1. Forberedelser <ol><li> For eggcelle kultur, kjøp eller fersk forberede Minimal Essential Medium (MEM) og supplere med 3 mg / ml bovint serumalbumin (BSA) som skissert i tabell 1. Plasser en polystyren flaske på en laste balanse (tare til null). Legg alle reagenser, med unntak av BSA, for å hente opp og sluttvolumet med MQ-vann på vektbasis, til totalt 250...

<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. 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Selv om det finnes flere metoder for eksogen nukleinsyre overføring inn i somatiske celler, som for eksempel elektroporering og transfeksjon, mikroinjeksjon er den optimale fremgangsmåten for levering av RNA-molekyler inn i transkripsjonelt hvilende muse-oocytter. Den nåværende protokollen gir en effektiv tilnærming for in vitro uttømming av spesifikke mRNA som muliggjør funksjonstesting av ulike spindel og / eller MTOC-tilknyttede faktorer i eggceller. Denne tilnærmingen resulterer i effektiv transkrip...

Meiose er en unik divisjon prosess som skjer i kjønnscellene (egg og sperm) og innebærer to delin uten å gripe DNA syntese å skille homologe kromosomer og søsterkromatider under meiose-I og meiose-II, henholdsvis 1. Feil i kromosom segregering under meiotisk divisjon i ubefruktede egg kan resultere i Aneuploidy, som er arvet av fosteret under befruktning. Spesielt var forekomsten av Aneuploidy i utviklings embryoer øker med stigende mors alder og er en viktig årsak til medfødte misdannelser samt graviditet tap på kvinner 1,2, altså, understreker et viktig behov for å forstå den molekylære grunnlaget for Aneuploidy under meiotisk divisjon . Under celledeling, er avgjørende avhengig montering av microtubule spindelapparatet og etablering av stabile kromosom-microtubule interaksjoner for riktig vedlegg til motsatt spindel kromosom segregeringpolene. Viktigere, meiotisk spindel formasjonen i pattedyr oocytter skiller seg fra mitose i somatiske celler, og er regulert av unike mikrotubul organiserende sentre (MTOCs) som mangler Sentrioler 3,4. Essensielle proteiner som er nødvendige for microtubule kimdannelse og organisering lokalisere til oocytten MTOCs, inkludert γ-tubulin som katalyserer microtubule montering. I tillegg pericentrin fungerer som en viktig stillas protein, som binder og ankere y-tubulin så vel som andre faktorer på MTOCs 5. Spesielt våre studier viser at uttynning av nøkkel MTOC-assosierte proteiner forstyrrer meiotisk spindel organisasjon og fører til kromosom segregering feil i ubefruktede egg, som ikke er fullt ut løses av spindelenheten sjekkpunkt (SAC) 6,7. Derfor mangler i spindel stabilitet, som ikke utløser meiotisk arrest, utgjøre en betydelig risiko i å bidra til Aneuploidy. Til tross for sin avgjørende rolle i spindelenheten og organisasjon, eggcelle MTOC protein komposisjon og funksjonen er fortsatt dårlig forstått. Å teste funksjonen av spesifikke target-proteiner i pattedyr oocytter er krevende, ettersom cellene blir transkripsjonelt hvilende kort tid før gjenopptakelse av meiose 8,9. Derfor preovulatoriske eggceller stole på mors mRNA butikker å gjenoppta meiose og støtte meiotisk divisjon samt de første cleavage divisjoner etter befruktning 10,11. Effekten av RNA-interferens (RNAi) mediert degradering av mRNA transkripter i pattedyr ubefruktede egg er godt etablert og mødre RNA rekruttert for oversettelse under meiotisk modning er spesielt mottagelig for siRNA målgruppe 12-14. Derfor mikroinjeksjon av korte interfererende RNA (siRNAs) inn oocytter gir en verdifull tilnærming til å utarme målet mRNAs for funksjonell testing. Her beskriver vi fremgangsmåter for isolering av muse-oocytter og siRNA-mediert nedbrytning av spesifikk transcripts å teste funksjonen av en essensiell MTOC-assosiert protein, pericentrin. I tillegg har vi beskrive immunfluorescens analyse forhold til å evaluere meiotisk spindel formasjonen i oocytter.

<table border="0">
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			<td></td>
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		</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>
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			<td>Major Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<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.

Mikroinjeksjon av sirnas gir en effektiv tilnærming for mRNA nedbrytning og påfølgende protein utarming i ubefruktede egg, som muliggjør effektiv og svært spesifikke funksjonstesting av ulike målgrupper faktorer in vitro. Deretter blir immunfluorescens brukes for spesifikk fenotype analyse, så vel som for å validere protein uttømming i siRNA-injiserte oocytter. I dagens eksempel fluorescerende merking av individuelle ubefruktede egg med DAPI sammen med anti-tubulin og anti-pericentrin antistoffer aktiv...

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 presenterer vi en protokoll for spesifikke siRNA-mediert mRNA uttømming fulgt av immunofluorescensanalyse å evaluere meiotisk spindelenheten og organisasjon i mus oocytter. Denne protokollen er egnet for in vitro uttømming av vitnemål og funksjonell vurdering av ulike spindel og / eller MTOC-tilknyttede faktorer i eggceller.

Meiotisk Spindel Vurdering i Mouse Oocytter av siRNA-mediert lyddemping

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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