Members of the Levy and Gatlin labs as well as colleagues in the Department of Molecular Biology offered helpful advice and discussions. Rebecca Heald provided support in the early stages of developing this protocol. This work was supported by the NIH/NIGMS (R15GM106318) and the American Cancer Society (RSG-15-035-01-DDC).

<ol>
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The authors have nothing to disclose.

Here is presented a novel method to study mechanisms of nuclear size regulation during X. laevis development. Developmental progression is associated with dramatic changes in cell physiology, metabolism, division rates, and migration, as well as alterations in the sizes of cells and intracellular structures. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. The X. laevis embryo extract and nuclear shrinking assay described here allow for the study of size regulatory mechanisms in an in vitro context, circumventing the complex signals and physical constraints of the in vivo system. Furthermore, the open nature of the extract allows for its facile biochemical manipulation.
Troubleshooting some steps of the protocol may be necessary. Problems with nuclear assembly and growth can result from poor quality eggs, incomplete demembranation of sperm nuclei, use of sperm nuclei at too high of a concentration, or incubating nuclear assembly reactions at too low of a temperature. Removal of endogenous embryonic nuclei from late embryo extract can be difficult due to the small size of these nuclei. This step in the protocol may require more than one round of centrifugation or the use of alternate rotors to completely remove all nuclei. Failure of nuclei to become smaller in the shrinking assay may be due to poor quality embryos, starting with nuclei that are too small, or too low of an incubation temperature. Poor quality eggs and embryos are indicated by a large proportion of white puffy activated eggs, unequal distribution of yolk and pigment granules, and eggs laid in strings. Spinning down fixed nuclei onto coverslips occasionally causes fragmentation of the nuclei, which can be avoided by decreasing the centrifugation speed or increasing the glycerol and sucrose concentrations in the nucleus cushion buffer. Troubleshooting antibody selection and dilution for immunofluorescence of nuclei is also often necessary. Critical steps within these protocols include: using high quality eggs and embryos, removing activated and lysed eggs and embryos, ensuring sperm nuclei are fully demembranated and appropriately diluted, monitoring nuclear assembly and growth, correctly staging embryos, completely removing endogenous embryonic nuclei from late stage embryo extracts, centrifuging fixed nuclei onto glass coverslips, and immunofluorescent staining of nuclei for visualization and quantification.
Nuclear size differences have long been noted between different species, cell types, disease states, and stages of embryonic development, indicating that nuclear size is tightly regulated1-10. Cancer of the prostate, breast, and ovary, to name a few, are all diagnosed and staged based on graded increases in nuclear size14,30-32. It is unclear to what extent changes in nuclear size are a cause or consequence of disease, and whether normal scaling of nuclear size contributes to proper development. Answering these functional questions necessitates an understanding of the mechanisms that control nuclear size. The assay presented here provides one approach to identifying novel pathways and proteins that regulate nuclear size, with potential relevance to cancer and development.
The Xenopus egg and embryo extract systems provide particularly powerful approaches to investigate mechanisms of organelle size regulation. While Xenopus extracts have been used for decades to study various aspects of cell cycle regulation and organelle assembly and dynamics, these extracts have more recently provided insights into size regulation of the nucleus3,23,33, mitotic spindle34-36, and chromosomes37-39. One of the major advantages of working with X. laevis embryo extracts is that they can be isolated from different stages of development, representing cytoplasm from differently sized cells containing nuclei of differing sizes. The method for investigating nuclear re-sizing presented here is physiologically relevant and offers a simplified system with which to identify mechanisms of nuclear size regulation. One complication of studying size regulation in vivo is that it is difficult to distinguish effects of spatial constraints and volume-limited components on size versus developmentally regulated changes in the expression or activities of size-controlling factors40. The approach described here focuses on developmentally controlled nuclear size regulators, using a robust nuclear re-sizing assay that is amenable to biochemical dissection.
Using the methods presented here, we identified protein kinase C (PKC) as a novel regulator of nuclear size. Furthermore, we showed that PKC activity and nuclear localization increase during X. laevis development23,41. A key limitation to the nuclear shrinking assay is that it is performed entirely in vitro. While Xenopus extract systems provide a simplified platform to discover new factors that regulate size, it is important that these results are validated in vivo. There are a variety of different approaches to corroborate physiological significance. X. laevis embryos can be microinjected with mRNA or morpholinos to alter the levels of nuclear scaling factors identified in extract, and then the effects on nuclear size can be quantified. Treatment of embryos with cell-permeable small molecule inhibitors or activators is also possible. For instance, we observed nuclear shrinking in live embryos treated with a phorbol ester, and this shrinking occurred within interphase without a requirement for nuclear envelope breakdown and passage through mitosis23. Another approach is to alter the levels of nuclear scaling factors in tissue culture cells to assess conservation of nuclear size control mechanisms outside of Xenopus.
What are the functional implications of altered nuclear size? One possibility is that nuclear size influences chromosome and chromatin organization, thereby altering gene expression. This idea can be directly tested as mechanisms of nuclear size regulation are further elucidated. Interestingly, PKC signaling and nuclear size are both routinely deregulated in cancer cells, often representing more aggressive metastatic disease13,14,42,43. Whether there is a direct relationship between deregulated PKC signaling and altered nuclear size in cancer remains to be shown. There is still much to be learned about nuclear size regulation, and the assay presented here, or variants of it, may still yield new information about mechanisms of nuclear size control. Furthermore, this assay could easily be adapted for the study of scaling mechanisms of other organelles and intracellular structures such as the mitotic spindle, endoplasmic reticulum, chromosomes, and Golgi, to name a few.

細胞小器官の大きさは、典型的には、細胞の大きさに比例し、これはおそらく最高のセルサイズ1-10との核の大きさのスケーリングのために文書化されています。これは、細胞と核の大きさの両方の劇的な減少は、多くの場合、11,12を観察している胚形成および細胞分化、中に特に当てはまります。また、変更された核の大きさは、癌の診断および予後13-17で重要なパラメータです。核のサイズの調節に寄与メカニズムは複雑さに起因する部分と核構造と機能の本質的な性質で、不明な点が多いです。ここで説明する方法は、生化学的操作および核のサイズ調節のメカニズムの解明に適している核のサイズスケーリングのためのin vitroアッセイとして開発されました。 アフリカツメガエルの卵抽出物は、in vitroでの複雑な細胞過程を再現し、研究するために十分に確立されたシステムであり、 </em&gt;コンテキスト。これらの抽出物は、アセンブリおよび紡錘体、小胞体の機能、および核18-22を含むいくつかの細胞プロセスに関する新たな基本的な情報を明らかにしました。抽出システムへの重要な利点の1つは、Xです。ツメガエル卵抽出物は、その組成が容易に組換えタンパク質または免疫除去の添加によって、例えば、変更することができ、ほぼ未希釈の細胞質を表します。また、一方がさもなければインビボの状況において致死的であるかもしれない治療を使用することによって本質的なプロセスを操作することができます。卵抽出物手順の変更は、Xからの抽出物の単離を可能にツメガエル胚ではなく、卵、およびこれらの胚抽出物は、生化学的な操作23にも同様に適しています。 X.中に数千50μを生成する-開発をアフリカツメガエル 、単細胞受精胚（〜1ミリメートルの直径）は12急速な細胞分裂（8ステージ1）のシリーズを受けますメートルの直径と発達段階に到達したより小さな細胞は、中期胞胚変移（MBT）またはステージ8 24-26と呼ばれます。 MBTは接合体、転写、細胞遊走、非同期細胞分裂、ギャップ相の取得、および核定常状態のサイズではなく、事前MBT胚のように継続的な原子力拡大の設立の発症によって特徴付けられます。ステージ4から原腸形成（段階10.5から12）に、個々の核の体積が10倍以上11により減少します。 ここでは、目標は、発達の進行の間の核の大きさで、これらの削減のための責任のメカニズムを識別することです。アプローチは、最初のXで核を組み立てることです卵抽出物をアフリカツメガエル卵細胞質/抽出物から、それらの核を単離すること。これらの核は、その後、後期原腸段階の胚から細胞質中に再懸濁されています。インキュベーション期間後、卵抽出物からの核は後期胚抽出物中より小さくなります。我々は、このwoulと推論しました発達核のサイズスケーリングに貢献後期胚に存在する細胞質成分を同定するために有用なアッセイをd。 インビボでの検証と結合し、このアッセイを用いて、我々は、プロテインキナーゼC（PKC）は、Xの核の大きさの発達削減に寄与することが実証され23を アフリカツメガエル 。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 568 Donkey anti-mouse IgG</td>
			<td>Molecular Probes</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP disodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Sigma Aldrich</td>
			<td>E1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>J2-21M</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge rotor</td>
			<td>Beckman</td>
			<td>JS 13.1</td>
			<td></td>
		</tr>
		<tr>
			<td>chymostatin</td>
			<td>Sigma Aldrich</td>
			<td>C7268</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphate disodium</td>
			<td>Calbiochem</td>
			<td>2380</td>
			<td></td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Sigma Aldrich</td>
			<td>C6255</td>
			<td></td>
		</tr>
		<tr>
			<td>cytochalasin D</td>
			<td>Sigma Aldrich</td>
			<td>C8273</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable wipes (kimwipes)</td>
			<td>Sigma Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>W326306</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass crystallizing dish (150x75 mm)</td>
			<td>VWR</td>
			<td>89090-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Macron</td>
			<td>5094-16</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst - bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>HCG - Human Chorionic Gonadotropin&#160;</td>
			<td>Prospec</td>
			<td>hor-250-c</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 Media</td>
			<td>Sigma Aldrich</td>
			<td>L4386</td>
			<td></td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma Aldrich</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysolecithin</td>
			<td>Sigma Aldrich</td>
			<td>L1381</td>
			<td></td>
		</tr>
		<tr>
			<td>mAb414</td>
			<td>Abcam</td>
			<td>ab24609</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>EMD</td>
			<td>MX0045-2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma Aldrich</td>
			<td>M9397</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Sigma Aldrich</td>
			<td>M5885</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>BDH</td>
			<td>56009</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin + Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>Pp0781</td>
			<td></td>
		</tr>
		<tr>
			<td>pepstatin</td>
			<td>Sigma Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic paraffin film (parafilm)</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Mallinckrodt</td>
			<td>70100</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Baker</td>
			<td>5 3140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG - Pregnant Mare Serum Gonadotropin</td>
			<td>Prospec</td>
			<td>hor-272-a</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher</td>
			<td>BP328</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>EMD</td>
			<td>SX0720-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>EMD</td>
			<td>SX0590</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle</td>
			<td>Thomas Scientific</td>
			<td>3411D56</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom glass tubes, 15 ml</td>
			<td>Corex</td>
			<td>8441</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG)</td>
			<td>ThermoFisher</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Calbiochem</td>
			<td>8550</td>
			<td></td>
		</tr>
		<tr>
			<td>thermal cycler</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>L8-80M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman</td>
			<td>SW 50.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield (anti-fade mounting medium)</td>
			<td>Vector</td>
			<td>H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation

細胞生物学の根本的な疑問は、細胞や細胞小器官のサイズが規制されている方法です。長い細胞や核のサイズの両方の劇的な削減が発生したときに、核の大きさは、一般的に胚形成の間、特に、セルの大きさに比例することが認められています。核のサイズ調節のメカニズムは不明な点が多いと変更された核の大きさがキー診断および予後のパラメータである癌に関連する可能性がある。 インビボ核サイズ調節因子が核機能の本質的かつ複雑な性質によって複雑されている識別に近づきます。核のサイズ制御を研究するためにここに記載のin vitroアプローチは、 アフリカツメガエルの開発中に発生する核の大きさが通常の減少を利用しています。まず、核はXに組み立てられます卵抽出物をアフリカツメガエル 。そして、これらの核は、後期胚から単離され、細胞質中に再懸濁します。 90分のインキュベーション期間、核の表面積 - 30後発達核のサイズスケーリングに貢献後期胚に存在する細胞質成分を識別するために有用なアッセイを提供し、60％ - 20によって減少します。このアプローチの主な利点は、卵や胚抽出物は、生化学的に核の大きさを調節する新規タンパク質や活動の同定を可能にする、操作することが可能な相対的な施設です。任意のインビトロのアプローチと同様に、in vivo系での結果の検証が重要であり、Xのマイクロインジェクションツメガエル胚は、これらの研究のために特に適しています。

卵抽出物中の核の組立 このプロトコルの最初のステップは、Xを準備していますツメガエルの卵抽出物（プロトコル1）および脱膜化精子核（プロトコル2）。これらの試薬 ​​は、その後、核デノボ （プロトコル3）を構築するために使用されている。1は、いくつかの代表的なデータを示しています。カルシウムの添加は、間期に減数分裂逮捕卵抽出物を駆動し、シクロヘキシミドは間期で逮捕された抽出物を保持します。 30で - 反応開始後45分で、最初は薄いS字型の精子核はスラグ状のクロマチン塊に厚くを開始する必要があります。無傷の核エンベロープの成功した形成は、GST-GFP-NLS（データは示していない）、またはFGリピートを認識するmAb414を用いた核膜孔複合体のためのリム染色などによる蛍光標識された輸入貨物の輸入及び保持によって評価することができますcontai寧ヌクレオポリン（ 図1A）。核アセンブリが発生した後、核の形状がより丸くなるべきと核エンベロープが拡大し、核タンパク質が（ 図1B-C）インポートされる核体積が増加するはずです。無傷の核エンベロープまたは核成長の形成に従わない場合には、品質の悪い卵抽出物を示しています。この場合には、卵の新たなバッチで再起動するのが最善の方法です。 胚芽抽出からの内因性核の除去 次のステップでは、後期胚抽出物（プロトコル4）を調製することです。最初の抽出物の調製及び前核の除去後は、抽出物の少量のヘキスト染色スカッシュを準備するのが最善です。多くの小さな胚の核の可視化は、抽出物（ 図2A）を製造するのに成功したの良好な指標です。内因性の胚の核は、下流analysiに干渉することができますようにSは、希釈し、遠心分離によって核を除去した後、抽出物のアリコートを確認することも重要です。最初のスカッシュと比較すると、非常に少数の核は、抽出物（ 図 2B）にしておく必要があります。現在、多くの核がまだある場合は、遠心分離の第二ラウンドが必要となります。 原子力縮小アッセイ 図3は、核の縮小アッセイ（プロトコル5）のための代表的な結果を示しています。後期胚抽出液中に再懸濁卵抽出物核は時間（ 図3B）の上に小さくなります。アッセイのための良好なネガティブコントロールは熱が不活性化された胚芽抽出物（ 図3A）で核をインキュベートすることです。核は熱不活性化エキスに収縮しないように、これは、未処理の胚抽出物で縮小核は浸透圧効果の結果ではありません観察いくつかの信頼性を提供します。核の大きさの程度観察された減少は、卵抽出物核の特定のバッチだけでなく、胚抽出物の品質に依存して変化します。我々の経験では、核の縮小は、常にしかし、別の核でこれらの実験を複数回繰り返すことが重要であり、アッセイの固有の変動に対処するために抽出し、（ 例えば、40種類以上の胚抽出物において）が観察される。4図は概略図を示していますプロトコル全体の。 <img alt="図1" src="/files/ftp_upload/54173/54173fig1.jpg" /> 図 X. 1.原子力組立時間コース ツメガエル 卵抽出。核はXで新たに組み立てました核膜孔複合体（NPC）（mAb414）でに対する抗体を用いた免疫蛍光法により、同じ反応からの3アリコートを固定して可視化した議定書に記載されているように卵抽出物をアフリカツメガエル ： <strong<html> &gt; A）30分、B）60分、及びC）の核アセンブリの開始後90分。免疫プロトコルは、スケールバーは25μmである。プロトコル5に記載されている<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54173/54173fig1large.jpg" target="_blank">。この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54173/54173fig2.jpg" /> X から核の 図2. 取り外し 胚芽抽出を アフリカツメガエル 。X. A）は、従来の遠心分離によって核を除去した後の核の除去、及びB）に：プロトコル4に記載の抽出物の少量のアリコートを固定し、ヘキストで染色した12胚- ツメガエル胚抽出物は、ステージから11.5を調製しました。スケールバーは25μmです。オム/ファイル/ ftp_upload / 54173 / 54173fig2large.jpg &quot;ターゲット=&quot; _空白&quot;&gt; この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図3" src="/files/ftp_upload/54173/54173fig3.jpg" /> 図3. 代表的なデータ原子力縮小アッセイから。核はXで新たに組み立てました組換えGFP-LB3を補っツメガエルの卵抽出物は、（核ラミナを可視化します）。内因性の胚の核が除去された12胚芽抽出 - プロトコル5で説明したように、卵抽出物の核を段階で11.5を単離し、そして再懸濁しました。ライブタイムラプスイメージングは​​、90分間、30秒間隔で行いました。 A）後期胚抽出物（LEE）、およびB）熱不活性化（HI）胚芽抽出物：図パネルは中でインキュベートし、核のための6分間隔で取得された画像を示します。時間のコースからの画像を続行します左から右から。スケールバーは25μmである。C）核は46の異なる卵と胚抽出物からのデータを縮小します。バーは&gt; 240の核のための手段を示しています。エラーバーは標準偏差である。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54173/54173fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54173/54173fig4.jpg" /> 原子力収縮アッセイの 図4 の回路図は、各色付きのボックスには、1プロトコルを表します。赤はXの準備を示していますツメガエルの卵抽出物（プロトコル1）。ブルーは、脱膜化Xの準備を示していますラエビスの精子（プロトコル2）。ピンク卵抽出物（プロトコル3）にデノボ核アセンブリを示しています。グリーンはXの準備を示していますツメガエル胚抽出物（プロトコル4）。紫は、核のを示していますhrinkingアッセイプロトコルおよび免疫蛍光（プロトコル5）。この図の一部は、23から再利用されている。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54173/54173fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> バッファ名 </td><td> 緩衝液組成 </td></tr><tr><td>マルクの改変リンガー（MMR）、1/3倍</td><td> 33のNaCl、0.7のKCl、0.3mMのMgSO 4を、0.7 mMのCaCl 2を、1.7 mMのHEPES、pHが7.4 </td></tr><tr><td>抽出バッファー（XB）、10× </td><td> 1 MのKCl、1mMのCaCl 2を、10mMのMgCl 2を、500mMのスクロース、100mMのHEPES、pHを7.8（pHを10 N KOHで調整し、4℃で保存） </td></tr><tr><td>緩衝液B </td><td>システインは冷たいのddH 2 Oを用いて調製0.8倍のXB（、pHがaで4％を溶かし10 N NaOHで7.8にdjusted） </td></tr><tr><td>緩衝液C </td><td>風邪のddH 2 Oの900ミリリットルで10倍XBの100メートルを混ぜます</td></tr><tr><td>緩衝液D </td><td>バッファCプラス5mMのEGTAおよび800μMのMgCl 2 </td></tr><tr><td> LPC株、1,000倍</td><td> 10 mg / mlでロイペプチン、ペプスタチン、およびDMSOに溶解したキモスタチンの各々は、（-20℃でアリコートで保存します） </td></tr><tr><td>緩衝液E </td><td> 100 mlのBuffer Dプラス100μlのLPCの株式</td></tr><tr><td>エネルギーミックス、50倍</td><td> 190 mMのクレアチンリン酸二ナトリウム、25mMのATP二ナトリウム塩、25mMの塩化マグネシウム</td></tr><tr><td>バッファT </td><td> 15mMのパイプ、15ミリモルのNaCl、5mMのEDTA、7ミリモルのMgCl 2、80mMのKClを、0.2Mのスクロース、pH7.4の（フィルタを滅菌し、4℃で保存） </td></tr><tr><td>バッファS </td><td>マルトースを20mMおよびバッファTで製造したリゾレシチン0.05％ </td></tr><tr><td>バッファR </td><td>バッファTプラス3％のウシ血清アルブミン</td></tr><tr><td>カルシウム原液、20倍</td><td> 10mMのCaCl 2を、0.1 MのKCl、1mMのMgCl 2（-20℃で保存） </td></tr><tr><td>核修正</td><td> 125μlの2 Mショ糖、12.5μlの1 M HEPES pHは7.9、250μlの37％ホルムアルデヒド、112μlののddH 2 O、0.5μlの10 mg / mlとヘキスト（2週間まで室温で保存） </td></tr><tr><td>修正されたバースの生理食塩水（MBS）、10倍</td><td> 880のNaCl、10mMの塩化カリウム、50mMのHEPES、25mMのNaHCO 3を、pHが7.8 </td></tr><tr><td>ハイソルトMBS </td><td> 1×MBSプラス0.7 mMのCaCl 2を、20 mMのNaClを</td></tr><tr><td>卵溶解緩衝液、ELB </td><td> 250mMのスクロース、50mMの塩化カリウム、2.5のMgCl 2、10mMのHEPES、pHは7.8 </td></tr><tr><td>核クッションバッファ</td><td> 1X XB、0.2 Mスクロース、25％グリセロール（濾過滅菌し、4℃で保存） </td></tr></tbody></table>表1バッファ・ページをther.within。このプロトコルに記載されたすべてのバッファの組成物は、この表に記載されています。

Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

用いた無細胞アッセイ<em&gt;アフリカツメガエル</em&gt;胚は、核サイズの規制のメカニズムを研究するために抽出します

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

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Biology

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

8.2K ビュー.  University of Wyoming. Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

ビデオ: A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

プラスチック製の埋め込みとアフリカツメガエル胚のセクショニング

生物学

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

アフリカツメガエル女性から卵子を入手

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

アフリカツメガエル卵抽出物の調製と分画

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

分別アフリカツメガエルの卵抽出を用いたin vitro核アセンブリで

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

アフリカツメガエル卵母細胞のマイクロインジェクション

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

でアニマルキャップの移植（ACT）アッセイを用いて組織の決定アフリカツメガエル</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

トランスジェニックの生産アフリカツメガエル制限酵素を介した統合と核移植によって

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

用いたDNA損傷チェックポイントの研究<em&gt;アフリカツメガエル</em&gt;卵抽出

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

の生産<em&gt;アフリカツメガエルトロピカ</em微小管関連RNAを識別するために、&gt;卵抽出

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

顔面移植<em&gt;アフリカツメガエル</em&gt;胚

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...