Name: isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders
Uploaded: 2016-12-05T12:30:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01 GM33397. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.G.G. is American Cancer Society Professor of Developmental Genetics.

<ol>
	<li>Purkinje, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Symbolae ad ovi avium historiam ante incubationem. , (1830).</li><li>R&#252;ckert, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Entwickelungsgeschichte+des+Ovarialeies+bei+Selachiern.">Zur Entwickelungsgeschichte des Ovarialeies bei Selachiern.</a> Anat. Anz. 7, 107-158 (1892).</li><li>Callan, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lampbrush Chromosomes. 36, (1986).</li><li>Gall, J. G., Callan, H. G., Wu, Z., Murphy, C., Kay, B. K., Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampbrush+chromosomes.">Lampbrush chromosomes.</a> Xenopus laevis: Practical Uses in Cell and Molecular Biology. Vol. 36 Methods in Cell Biology , 149-166 (1991).</li><li>Gall, J. G., Wu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+contents+of+isolated+Xenopus+germinal+vesicles.">Examining the contents of isolated Xenopus germinal vesicles.</a> Methods. 51, 45-51 (2010).</li><li>Penrad-Mobayed, M., Kanhoush, R., Perrin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tips+and+tricks+for+preparing+lampbrush+chromosome+spreads+from+Xenopus+tropicalis+oocytes.">Tips and tricks for preparing lampbrush chromosome spreads from Xenopus tropicalis oocytes.</a> Methods. 51, 37-44 (2010).</li><li>Morgan, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+oocyte+nuclei:+cytological+preparations+of+active+chromatin+and+nuclear+bodies+from+amphibian+germinal+vesicles.">Working with oocyte nuclei: cytological preparations of active chromatin and nuclear bodies from amphibian germinal vesicles.</a> Methods Mol. Biol. 463, 55-66 (2008).</li><li>Paine, P. L., Johnson, M. E., Lau, Y. T., Tluczek, L. J., Miller, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oocyte+nucleus+isolated+in+oil+retains+in+vivo+structure+and+functions.">The oocyte nucleus isolated in oil retains in vivo structure and functions.</a> Biotechniques. 13, 238-246 (1992).</li><li>Handwerger, K. E., Cordero, J. A., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cajal+bodies,+nucleoli,+and+speckles+in+the+Xenopus+oocyte+nucleus+have+a+low-density,+sponge-like+structure.">Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure.</a> Mol Biol Cell. 16, 202-211 (2005).</li><li>Patel, S., Novikova, N., Beenders, B., Austin, C., Bellini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+images+of+RNA+polymerase+II+transcription+units.">Live images of RNA polymerase II transcription units.</a> Chromosome Res. 16, 223-232 (2008).</li><li>Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octagonal+nuclear+pores.">Octagonal nuclear pores.</a> J Cell Biol. 32, 391-399 (1967).</li><li>L&#246;schberger, A., 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=Super-resolution+imaging+visualizes+the+eightfold+symmetry+of+gp210+proteins+around+the+nuclear+pore+complex+and+resolves+the+central+channel+with+nanometer+resolution.">Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution.</a> J Cell Sci. 125, 570-575 (2012).</li><li>Gottfert, F., 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=Coaligned+dual-channel+STED+nanoscopy+and+molecular+diffusion+analysis+at+20+nm+resolution.">Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution.</a> Biophys J. 105, L01-L03 (2013).</li><li>Wallace, R. A., Jared, D. W., Dumont, J. N., Sega, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+incorporation+by+isolated+amphibian+oocytes:+III.+Optimum+incubation+conditions.">Protein incorporation by isolated amphibian oocytes: III. Optimum incubation conditions.</a> J. Exp. Zool. 184, 321-333 (1973).</li><li>Bridger, J., Spector, D. L., Goldman, R. D., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situhybridization+to+DNA.">Fluorescence in situhybridization to DNA.</a> Cells: A Laboratory Manual. 3, 111.111-111.136 (1998).</li><li>Singer, R. H., Spector, D. L., Goldman, R. D., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+to+RNA.">In situ hybridization to RNA.</a> Cells: A Laboratory Manual. 3, 111-116 (1998).</li><li>Gardner, E. J., Nizami, Z. F., Talbot, C. C., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+intronic+sequence+RNA+(sisRNA),+a+new+class+of+noncoding+RNA+from+the+oocyte+nucleus+of+Xenopus+tropicalis.">Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis.</a> Genes Dev. 26, 2550-2559 (2012).</li><li>Talhouarne, G. J., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lariat+intronic+RNAs+in+the+cytoplasm+of+Xenopus+tropicalis+oocytes.">Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.</a> RNA. 20, 1476-1487 (2014).</li><li>Duryee, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+nuclei+and+non-mitotic+chromosome+pairs+from+frog+eggs.">Isolation of nuclei and non-mitotic chromosome pairs from frog eggs.</a> Arch. Exp. Zellforsch. 19, 171-176 (1937).</li></ol>

The authors have nothing to disclose.

カエルやサンショウウオの手孤立のGVから「生きている」のLBCsの最初の観測は、蛍光免疫染色の前に、およびFISHの前に、アメリカの生物学者ウィリアム・Duryee、位相コントラストやDIC顕微鏡の導入前の19で、ほぼ80年前に作られました。リアルな方法でそれらを修正するために、その基本的な形態を破壊することなく、分子技術を適用するために、ガラススライドにのLBCsを取り付け?...

ほとんどの脊椎動物は、有袋類および胎盤哺乳類の注目すべき例外を除いて、大規模な卵黄のような卵を産みます。その時々巨大なサイズにもかかわらず、これらの卵はまだ女性の卵巣ながら、最終寸法に達する単一細胞です。卵巣卵は卵母細胞と呼ばれ、それぞれは、典型的には、単に胚胞またはGV早ければ19 世紀から知られている単一の巨大な核を、含まれています。一般的な実験のカエル、 アフリカツメガエルやアフリカツメガエルトロピカの1卵母細胞は、それぞれ1.2ミリメートルと0.8ミリメートルの最大直径（ 図1）に達します。直径0.4ミリメートル（ 図2、図3） -これら2カエルの成熟卵母細胞からのGVは0.3です。サンショウウオは、典型的には、さらに大きく卵母細胞とのGVを持っています。メキシコアホロートル、Ambystomaのmexicanumの完全に成熟卵母細胞は、直径2 mm以上であり、GVは約0.5mmです。したがって、これらの核は、肉眼や缶に容易に表示されます典型的な体細胞の核では不可能であり、多くの方法で操作すること。 同様に顕著GV内の染色体の巨大なサイズ、19 世紀の終わりに既に認識された事実です。 Ambystomaおよび他のサンショウウオの個々の染色体の長さは最大1ミリメートル（ 図4、図5）とすることができます。 100μm以上までの長さで、彼らはほとんどの生物の典型的な体細胞の染色体を矮星が、 アフリカツメガエルのものは、小さいかなりあります。対の横方向のループの数百（ 図5） -卵母細胞染色体の重要な特徴は、それらの最も特徴的な形態学的特徴の一つにつながり、それらの異常な転写活性です。各ループは積極的にRNAを合成する1つまたは少数の転写単位から構成されています。ループは、卵母細胞は、その表面的な後に名前 &quot;ランプブラシ&quot;染色体につながったファジーな外観を、染色体与えます灯油ランプの煙突をきれいにするために、以前の回で使用したブラシに似ています。 2 この論文の焦点はのLBCs及び核細胞小器官（核小体、ヒストン遺伝子座体、およびスペックルを）勉強する孤立のGVの使用にあります。二つかなり異なる技術について説明します。まず、より一般的な技術では、のGVを簡単接着卵黄を除去するためにリンス、宝石商の鉗子を使用して、食塩水で単離される、および核膜を宝石商の鉗子で再び除去されます。 LBCsと核細胞小器官を含むゲル状の内容は、顕微鏡スライドガラスまたはカバーガラス上に定着させています。このような製剤は、位相コントラストやDIC顕微鏡によって直接調べることができます。また、調製物は、スライドまたはカバースリップにのLBCsや細胞小器官を添付するために遠心分離することができます。このような調製物は、その後、主に免疫蛍光及びfluorescenによって、核酸およびタンパク質の詳細な分子分析のために処理することができますin situハイブリダイゼーション（FISH） における T。 3-7 第2の技術は、鉱油中のGVの単離を含みます。 8オイル分離されたのGVは、多くの時間のための転写活性のままで、もう1つは核の内容は、できるだけ実物そっくりになりたいと思っている研究に有用である可能性があります。 9,10核「樹液」の屈折率はのLBCsと他の核内小器官（ 図3）に近いので、顕微鏡の技術は、油分離されたのGVと挑戦することができます。 最後に、それらの大きさや操作のしやすさの、のGVは核膜の研究のための理想的な材料です。核膜孔複合体は、最初の両生類のGVエンベロープ11とより最近の超解像観察に電子顕微鏡研究から説明したのと同じ材料を使用しています。 12,13

<table><tbody><tr><td>Paraformaldehyde (reagent grade, crystalline)</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>Despite warnings in many protocols, a concentrated solution can be stored indefinitely at RT</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma-Aldrich</td><td>A5040-100G</td><td>Sometimes referred to as MS-222</td></tr><tr><td>Ethicon RB-1 1/2 circle taper point 3-0 sutures</td><td>VWR&amp;nbsp;</td><td>95057-000</td><td></td></tr><tr><td>Paraplast (paraffin wax)</td><td>Sigma-Aldrich</td><td>P3558-1KG</td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Phenylenediamine</td><td>Sigma-Aldrich</td><td>P6001</td><td></td></tr><tr><td>Gelatin</td><td>Grocery Store</td><td></td><td>Commercial Knox gelatin works fine</td></tr><tr><td>ProLong Gold antifade mountant</td><td>ThermoFisher Scientific</td><td>P10144</td><td></td></tr><tr><td>Gold Seal cover glass&amp;nbsp; 22 x 22 mm #1 1/2 (0.16 - 0.19 mm thick)</td><td>Electron Microscopy Sciences</td><td>63786-01</td><td>These coverslips are the recommended thickness for superresolution microscopy</td></tr><tr><td>Dumont forceps #5</td><td>Electron Microscopy Sciences</td><td>72700-D</td><td>http://www.emsdiasum.com/microscopy/products/tweezers/dumont_positive_action.aspx</td></tr><tr><td>Paraffin oil (light)&amp;nbsp;</td><td>EMD Chemicals</td><td>PX0047-1</td><td>For isolating GVs in oil</td></tr><tr><td>Adhesive &lt;em&gt;in situ&lt;/em&gt; PCR and hybridization chambers (25 &amp;micro;L)</td><td>BioRad</td><td>Frame-Seal Slide Chambers #SLF0201</td><td>http://www.bio-rad.com/en-us/sku/slf0201-frame-seal-slide-chambers</td></tr><tr><td>Silicone isolators</td><td>Grace-Biolabs</td><td>select from catalog link</td><td>http://www.gracebio.com/life-science-products/microfluidics/silicone-isolators.html</td></tr><tr><td>Coplin jars and staining dishes</td><td>Electron Microscopy Sciences</td><td>select from catalog link</td><td>http://www.emsdiasum.com/microscopy/products/histology/staining.aspx</td></tr></tbody></table>

isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders

私たちは、カエルやサンショウウオの卵母細胞、または埋葬されていない卵、で見つかった巨大な転写活性ランプブラシ染色体（のLBCs）を研究するための方法が記載されています。最大直径0.5mmに、自身の個々のLBCsの長さは、最大1ミリメートルとすることができ、彼らは巨大な核内に存在します。染色体のサイズが大きい光光学顕微鏡による活性遺伝子の比類のない観測を可能にするが、同時に、特別な技術は、核の単離核エンベロープを除去し、顕微鏡スライド上に染色体を拡散するために必要とされます。また、卵核胞（GV）と呼ばれる卵母細胞の核は、核アクチンの部分的なゲル化を可能にし、のLBCsの繊細な構造を保存媒体に隔離されています。このステップは、宝石商の鉗子を使用して、解剖顕微鏡下で手動で行われます。次に、核膜は、宝石商の鉗子で再び手動で削除されます。核内容はすぐにdispe媒体に転写されていますアクチンゲルRSESと損傷していないのLBCsは、顕微鏡スライド上に決済することができます。細かい詳細はブラウン運動によって隠されているが、この時点でのLBCs及び他の核小器官は、位相コントラストまたは微分干渉コントラスト顕微鏡で観察することができます。高解像度の顕微鏡観察や分子分析のために、製剤全体がスライドにしっかりと繊細なのLBCsを添付するために遠心分離されます。パラホルムアルデヒド中で簡単に固定が、その後免疫蛍光染色によって、またはin situハイブリダイゼーションに続いています。 LBCsは、転写活性状態にあり、それらの巨大なサイズは、共焦点又は超解像顕微鏡を使用して、個々の遺伝子レベルでの分子の分析を可能にします。

巨大なランプブラシ染色体を調べるために1は、カエルやサンショウウオの卵母細胞を単離することによって開始します。 図1はカエル、 アフリカツメガエルの卵巣から除去した後の緩衝溶液中で成熟した卵母細胞の集団を示しています。このような卵母細胞は、室温で日間良好な状態で残っています。核（または卵核胞）を、食塩水（ 図2）...

Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

私たちは、カエルやサンショウウオの卵母細胞を生きているから巨大な転写活性ランプブラシ染色体を単離するための簡単なテクニックを紹介します。私たちは、位相コントラストまたは微分干渉コントラスト、およびどのようにin situハイブリダイゼーションまたは免疫蛍光染色における蛍光のためにそれらを修正することにより、「生きている」これらの染色体を観察する方法について説明します。

カエルとサンショウウオのリビング卵母細胞からジャイアントランプブラシ染色体の分離

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and ...

isolation-giant-lampbrush-chromosomes-from-living-oocytes-frogs

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Genetics

Unit 1

DNA and Chromosome Structure

SCIENTISTS IN ACTION

12.9K ビュー.  Carnegie Institution for Science. 私たちは、カエルやサンショウウオの卵母細胞を生きているから巨大な転写活性ランプブラシ染色体を単離するための簡単なテクニックを紹介します。私たちは、位相コントラストまたは微分干渉コントラスト、およびどのようにin situハイブリダイゼーションまたは免疫蛍光染色における蛍光のためにそれらを修正することにより、「生き?...

ビデオ: Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作と<em&gt;インビトロ</emの&gt;成熟<em&gt;アフリカツメガエル</em細胞質内精子注入し&gt;卵母細胞、胚発生を研究するために

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

発生生物学

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

携帯無料ツメガエル卵抽出液のマイクロエマル ジョンにおける細胞周期振動の再構成

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

生化学

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

からの同時プリとポストシナプスの電気生理学的記録<em&gt;アフリカツメガエル</em&gt;神経筋共培養

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

神経科学

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

の受精アフリカツメガエル卵母細胞ホストの転送方式を使用して

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

生物学

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

生細胞イメージングとの胚上皮細胞の定量分析アフリカツメガエル</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgf&#223; family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgf&#223; receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgf&#223; precursor proteins. RNA encoding Tgf&#223; precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of&#160;gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgf&#223; cleavage products under physiologic conditions.

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

When Transforming Growth Factor &#223; family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

ゼノプス・ラエビス胚の芽球に分泌される成長因子ßファミリー切断産物の分析

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

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 ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、 アフリカツメガエル

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

無細胞アッセイは、有糸分裂の終了時クロマチン脱凝縮を勉強します

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

カエルとサンショウウオのリビング卵母細胞からジャイアントランプブラシ染色体の分離

要約

さらに動画を探す

この動画の章

操作と

携帯無料ツメガエル卵抽出液のマイクロエマルジョンにおける細胞周期振動の再構成

からの同時プリとポストシナプスの電気生理学的記録

の受精アフリカツメガエル卵母細胞ホストの転送方式を使用して

生細胞イメージングとの胚上皮細胞の定量分析アフリカツメガエル

ゼノプス・ラエビス胚の芽球に分泌される成長因子ßファミリー切断産物の分析

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

の生産

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、アフリカツメガエル

無細胞アッセイは、有糸分裂の終了時クロマチン脱凝縮を勉強します

カエルとサンショウウオのリビング卵母細胞からジャイアントランプブラシ染色体の分離

要約

さらに動画を探す

この動画の章

操作と

携帯無料ツメガエル卵抽出液のマイクロエマル ジョンにおける細胞周期振動の再構成

からの同時プリとポストシナプスの電気生理学的記録

の受精アフリカツメガエル卵母細胞ホストの転送方式を使用して

生細胞イメージングとの胚上皮細胞の定量分析アフリカツメガエル

ゼノプス・ラエビス胚の芽球に分泌される成長因子ßファミリー切断産物の分析

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

の生産

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、 アフリカツメガエル

無細胞アッセイは、有糸分裂の終了時クロマチン脱凝縮を勉強します

携帯無料ツメガエル卵抽出液のマイクロエマルジョンにおける細胞周期振動の再構成

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、アフリカツメガエル