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.

このプロトコルは、ジョージア大学の施設内動物管理使用委員会（IACUC）によって承認されました。 1.準備<ol><li>卵母細胞培養、購入やたての最小必須培地（MEM）を調製し、 表1で説明したように3 mg / mlのウシ血清アルブミン（BSA）で補うために。ロードバランス（ゼロに風袋）にポリスチレンボトルを置きます。ためには、BSAを除き、全ての試薬を追加し?...

<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. 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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. 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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>

そのようなエレクトロポレーション及びトランスフェクションのような体細胞への外因性核酸の転写のための複数の方法があるが、マイクロインジェクションは、転写休止マウス卵母細胞へのRNA分子の送達のための最適な方法です。現在のプロトコルは、異なる主軸および/ ​​または卵母細胞におけるMTOC関連因子の機能テストを可能にする特定のmRNAのインビトロ枯渇のための効果的...

減数分裂は、配偶子（卵母細胞および精子）で発生する独特の分割処理であり、それぞれ1、減数分裂Iと減数分裂IIの間に相同染色体と姉妹染色分体を分離するためにDNA合成を介在せずに2つの連続した部門が関与します。卵母細胞における減数分裂時の染色体分離におけるエラーは受精胚中に継承され異数性をもたらすことができます。特に、発展途上胚における異数性の発生率は、母体の加齢に伴って増加し、女性1,2における妊娠損失だけでなく、先天性の先天性欠損症の主な原因である、したがって、減数分裂時の異数性の分子基盤を理解するための重要な必要性を強調。 細胞分裂の間、染色体分離は反対スピンドルへの正しい取り付けのための微小管スピンドル装置と安定した染色体微小管の相互作用の確立の組み立てに決定的に依存していますポール。重要なことは、哺乳動物の卵母細胞における減数分裂紡錘体の形成は、体細胞での有糸分裂とは異なり、中心小体3,4を欠いているユニークな微小管組織化センター（MTOCs）によって規制されています。微小管の核形成と組織のために必要不可欠なタンパク質は、微小管集合を触媒するγチューブリンを含む卵母細胞MTOCs、に局在します。また、MTOCs 5でバインド必須の足場タンパク質として機能し、アンカーγチューブリン、ならびにその他の要因ペリセントリン。注目すべきことに、我々の研究は、キーMTOC関連タンパク質の枯渇が減数分裂紡錘体の組織を破壊し、完全に紡錘体チェックポイント（SAC）6,7によって解決されていない卵母細胞における染色体分離のエラーにつながることを示しています。したがって、減数分裂の停止を誘発しないスピンドル安定性の欠陥は、異数性に貢献において重要なリスクをもたらします。スピンドルアセンブリと組織内での重要な役割、卵母細胞MTOC protのにもかかわらず、EIN組成と機能が十分に理解されたままになります。 細胞が減数分裂まもなく8,9の再開前に、転写的に静止してしまうなどの哺乳動物の卵母細胞に特異的な標的タンパク質の機能をテストすることは、困難です。したがって、排卵前の卵母細胞が減数分裂を再開し、受精10,11の後に減数分裂と同様に最初の切断部門をサポートするために母性mRNAの店に依存しています。哺乳動物の卵母細胞中のmRNA転写物のRNA干渉（RNAi）介在性分解の有効性は、十分に確立され、減数分裂成熟の間の翻訳のために募集母性RNAは、siRNAが12-14を標的に特に適しています。したがって、卵母細胞への短い干渉RNA（siRNA）のマイクロインジェクションは、機能テストのために標的mRNAを枯渇させる貴重なアプローチを提供します。 ここでは、マウス卵母細胞及び特定transcriのsiRNA媒介枯渇を単離するための方法を記載PTSはペリセントリン、本質的​​なMTOC関連タンパク質の機能をテストします。加えて、我々は、卵母細胞における減数分裂紡錘体形成を評価するために免疫蛍光分析条件を記述する。

<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.

siRNAのマイクロインジェクションは、in vitroで異なるターゲット因子の効率的かつ高度に特異的な機能テストを可能にした卵母細胞におけるmRNAの分解及びその後のタンパク質の枯渇に有効なアプローチを提供します。その後、免疫蛍光は、特定の表現型分析のために使用されるだけでなく、siRNAを注入した卵母細胞中のタンパク質の枯渇を検証します。現在の例では、抗チューブリン?...

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

ここでは、マウス卵母細胞で減数分裂スピンドルアセンブリと組織を評価するために、免疫蛍光分析に続いて、特定のsiRNA媒介mRNAの枯渇のためのプロトコルを提示します。このプロトコルは、転写物のin vitroでの枯渇と異なるスピンドルおよび/ ​​または卵母細胞におけるMTOC関連因子の機能評価に適しています。

siRNA媒介サイレンシングによるマウス卵母細胞における減数分裂紡錘体の評価

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

Research

JoVE Journal

Biology

Nuclear Transfer into Mouse Oocytes

マウス卵母細胞への核移転

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Retrieval of Mouse Oocytes

マウス卵母細胞の検索

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

コレクションとハムスターの卵母細胞とマウス胚の凍結保存

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Flow Cytometry Purification of Mouse Meiotic Cells

マウス減数分裂細胞のフローサイトメトリーの精製

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

siRNAを介した遺伝子サイレンシングと組み合わせることで生細胞のカルシウムイメージングでは、CAを識別 2 + ER膜のリークチャネルとその調節機序

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

コットンでアグロバクテリウムを介したウイルス誘発遺伝子サイレンシングアッセイ

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

カチオン性リポソームを介した特異的siRNAの再配達によるマウスの肺におけるIntersectin-1Sの長期サイレン。電子顕微鏡によるノックダウン効果の評価

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...