We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.

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(45), (2010).</li><li>Ishibashi, S., Cliffe, R., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+bi-allelic+mutation+rates+using+TALENs+in+Xenopus+tropicalis).">Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis).</a> Biology open. 1 (12), 1273-1276 (2012).</li><li>Sakane, Y., 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=Targeted+mutagenesis+of+multiple+and+paralogous+genes+in+Xenopus+laevis+using+two+pairs+of+transcription+activator-like+effector+nucleases.">Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.</a> Development, growth &amp; differentiation. 56 (1), 108-114 (2014).</li><li>Nakayama, T., 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=Simple+and+efficient+CRISPR/Cas9-mediated+targeted+mutagenesis+in+Xenopus+tropicalis.">Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.</a> Genesis. 51 (12), 835-843 (2013).</li><li>Guo, X., 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=Efficient+RNA/Cas9-mediated+genome+editing+in+Xenopus+tropicalis.">Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.</a> Development. 141 (3), 707-714 (2014).</li></ol>

The authors have nothing to disclose.

我々ここで詳細母体要因を枯渇させるか、受精前に外因性因子を過剰発現する新しい方法。このシステムは、2マイクロインジェクションが必要ですが、その代わりにホスト転送方式7,17で使用されるカエルの手術を、スキップします。それは動物のケアの面でカエル手術手順を削除することが最適である。また、私たちは実験の成功に影響を与える一つの生物学的要因を取り除くことができます。つまり、転送実験の​​ためのホストの女性カエルの品質のために考慮することがありません。したがって、このシステムでPMSGプライムカエルから得た卵母細胞の品質が成功した実験のための鍵である主要な生物学的な要因である。卵母細胞の質は、卵巣の採取後またはdefolliculation後に判定することができる。異常がこれらの段階で観察される場合は、新しいカエルから別の卵巣を収集するために最適である。あなたは卵母細胞の品質を確認することができますもう一つのポイントは、卵成熟の後である。プロゲステロンの80％未満の場合電子処理された卵母細胞の成熟の兆候を示しては、さらなる分析のための胚の十分な数を得ることができないかもしれない。代わりに手動defolliculationの酵素defolliculationの使用もdefolliculationに要する労力を軽減するために、このシステムを使用する別の利点である。しかし、それは手動defolliculationが酵素処理よりも良い発展を与えることがまだ可能である。 図3に示されるように、 インビトロ成熟卵母細胞のほぼ10％が水泳幼生段階に到達することができる。これらのデータは、8、新しい注入実験で報告されたものを含む13の独立した実験から収集される。ホスト転送方法は、転送された卵母細胞の30〜60％が神経胚の段階17に到達することができるので、意味のあるデータを得るための各処理に75-150卵母細胞を必要とする。私たちの方法は、通常、切断された胚の約40から60まで%が到達することができるので、各実験群の200〜300卵母細胞で始まる筋肉の応答段階。これらのデータは、両方の方法は、合理的な発育速度をサポートすることを示唆している。私たちは、これまでのところ、このアプローチは変態による開発をサポートしていることを示す、この技術を用いて制御するmRNAを注入したと制御、アンチセンスオリゴ注入した卵母細胞から10生きているカエルを取得しています。 私たちの卵母細胞操作-ICSI法がすぐに受精後、非常に初期の胚発生時に母体の因子の役割をテストする機会を提供します。また、受精前のクロマチン修飾因子の過剰発現により、開発に必要な母性クロマチンの状態を理解するための受精の前に母方のクロマチンを改造することがあります。これらがあっても受精前に表現することができるので、この戦略はまた、そのような転写活性化因子のようなエフェクターヌクレアーゼ（TALEN）18,19およびCRISPR / CAS9 20,21として、現在の遺伝子編集システムでうまく動作する可能性があります。そのため、私たちの新しいアプローチがpotentiを持ってらは、将来的に多くの用途に使用される。

アフリカツメガエルは、開発1を研究するために広く使用され、強力なモデル生物である。 アフリカツメガエルの卵と豊富な（哺乳類のカウンターパートとの比較では約0.1ミリメートルであることに、約1.2〜1.4ミリメートル）異常 ​​に大きいためである。卵は（4,000-8,000細胞と段階8〜8.5で発生する、MBT）半ば胞胚遷移するまで、胚発生を駆動するのに十分である、母性を合成して記憶コンポーネントが含まれています。 MBTは、その後、さらなる開発を導く胚性遺伝子産物を産生する接合子ゲノム活性化を伴う。 開発のための重要な母性因子を同定することを目的とした多くの研究。多くの研究は、母体タンパク質の場合の劣化が腸胚段階2-4で観察することができる受精胚へのモルホリノオリゴヌクレオチドを含む、アンチセンスオリゴヌクレオチド（オリゴ）の注射に依存する。別の方法として、mRNAは受精し、EMに注入される遺伝子機能を邪魔したり、過剰に発現するタンパク質の運命を追跡するbryos。しかし、1細胞期胚への注入は通常、MBT前に非常に初期胚の段階で母親のタンパク質の発現レベルに影響を与えません。 Heasman氏とワイリーは、この問題5を克服するためにホスト転送方法を確立した。それらの方法では、手動で濾胞除去した卵母細胞は、アンチセンスオリゴを注射された宿主の雌6に移した。初期胚発生中のダウンレギュレートのタンパク質の役割を調べることができるように、タンパク質は、受精の前にダウンレギュレートされる。 7に総説として、この母体枯渇法は、母体タンパク質のいくつかのユニークな発達の役割が同定された。 本稿では、詳細受精前のmRNAの母親の枯渇や過剰発現が手動defolliculationとホスト転送せずに達成されている私たちの最近開発された方法、8。マニュアルdefolliculation、時間とホスト転送の多くは、多くの場合、このように母親の枯渇方法を頻繁に使用を妨げる、熟練した技術や動物の手術のため特定のライセンスを必要としますが必要です。 Defolliculationとホスト転送は、それぞれ、酵素defolliculation 9,10及び細胞質内精子注入（ICSI）11-13によって置換されている。卵へのICSIはもともとトランスジェニックカエル12を製造した。精子と胚の核はまた、インビトロ成熟両生類卵母細胞11,14,15に移植した。ここで、我々 は、in vitroに精子を注入するステップバイステップの方法は、アンチセンスオリゴまたはmRNAで予め注入された卵母細胞を成熟示す。

<table border="0" cellpadding="0" cellspacing="0" width="589">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pregnant Mare Serum Gonadotropin (PMSG)&#160;</td>
			<td style="width:87px;">MSD Animal Health</td>
			<td style="width:119px;">Vm 01708/4309</td>
			<td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>For anesthesia</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Liberase TM Research Grade</td>
			<td style="width:87px;">Roche</td>
			<td style="width:119px;">05 401 127 001</td>
			<td>For oocyte defolliculation. Store at -20oC</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:216px;">Drummond Nanoject</td>
			<td style="width:87px;">Drummond Scientific Company</td>
			<td style="width:119px;">3-000-205/206</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glass capillary&#160;</td>
			<td style="width:87px;">Alpha laboratories</td>
			<td style="width:119px;">7&#8221; Drummond #3-000-203-G/XL</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Puller&#160;</td>
			<td style="width:87px;">SUTTER Instrument</td>
			<td style="width:119px;">Model D-97</td>
			<td>For microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraPure Agarose</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td>For invitro maturation and ICSI</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">14 ml ultra-clear centrifuge tube&#160;</td>
			<td style="width:87px;">Beckman Coulter United Kingdom Ltd</td>
			<td style="width:119px;">344060</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">OptiPrep Density Gradient Medium (Iodixanol)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D1556</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digitonin</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D141</td>
			<td>Cell permeabilization reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaker</td>
			<td style="width:87px;">Hybaid&#160;</td>
			<td style="width:119px;">HB-SHK-1</td>
			<td>For oocyte defolliculation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope (Stereo zoom microscope)</td>
			<td style="width:87px;">ZEISS</td>
			<td style="width:119px;">Semi SV6</td>
			<td>For oocyte collection and microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 &#956;m pore filter</td>
			<td style="width:87px;">CellTrics</td>
			<td style="width:119px;">04-0042-2317</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultracentrifuge</td>
			<td style="width:87px;">Beckman Coulter</td>
			<td style="width:119px;">Optima L-100XP</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50 ml centrifuge tube</td>
			<td style="width:87px;">Cellstar Greiner Bio-One</td>
			<td style="width:119px;">227261 or 210261</td>
			<td>Oocyte collection and defolliculation reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml centrifuge tube</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">352097</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">90 mm Petri dish&#160;</td>
			<td style="width:87px;">Thermo Scientific</td>
			<td style="width:119px;">101VR20/C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 60x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">353004</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 35x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">351008</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

体外成熟卵子に使用してICSI胚の胚発生は、（ 図3A）を検討した。 MII段階へのGV卵母細胞の成熟率が可変であり、大部分は、卵母細胞の品質に依存します。良い実験では、GV卵母細胞のほぼ100％が最終的にMII段階で卵になって、卵成熟のプロゲステロンとショー兆候に対応する。全て成熟卵母細胞は、ICSIに供したと注入された卵の約25％が切断された（ 図3A、N =制御のための13は、無オリゴ注入した卵母細胞のためのオリゴ注入した卵母細胞であり、n = 7スクランブル）。約60％または対照オリゴ注入した卵母細胞または非注射卵母細胞から産生される切断された胚の80％が、それぞれ、胞胚/原腸胚段階に達した。胞胚/原腸胚の82パーセントがnでは良い品質のものであったのに対し、胞胚/原腸胚の中では、オリゴ注入されたサンプル中の胚の約半分は、（異常な切断およびアポトーシスのほとんどない兆候）良い品質のものであったに注入された試料（ 図3A）。非注射サンプル中の60％と、切断された胚の29％が（ 図3A）でしたが最後に、オリゴ注入されたサンプル中の41％と、切断された胚の11％が、それぞれ、筋肉の応答、スイミングオタマジャクシの段階に達した。これらICSI胚は、正常および異常な胚（ 図3B）の混合物である。そのうちのいくつかは変態を受け、カエル8成熟した開発する。これらの結果は、IVMとICSI続いGVを卵母細胞へのアンチセンスオリゴヌクレオチドの注入は、効率的な初期胚発生を可能にすることを示唆している。アンチセンスオリゴの注入自体が胚発生を減少するが、我々はまだようにそのような発達速度をチェックするなど、多くの実験目的のために十分な胚、RT-PCR、ウェスタンブロットとを取得することができます。 <img alt="図1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> イチジク<html> URE 1： アフリカツメガエルの典型的な例は、 体外成熟実験のために良い品質か悪い品質のツメガエル卵母細胞。 （A）悪い品質卵母細胞の例。例えば、斑状の色素沈着を示す卵母細胞は、その後の実験に使用されていない。各アフリカツメガエル卵母細胞は、直径が約1.2〜1.4ミリメートルであるツメガエル 。（B）部分的に濾胞除去卵母細胞。卵母細胞の右半分は、血管の存在によって識別され得る包細胞で覆われている。矢印は毛包細胞を含まず、注射針が注入されるに領域を示している。（C）等しいサイズであり、動物の脳半球および植物半球の間に明確なコントラストを均一着色された動物の半球を示す良質の卵母細胞の例。 2496 / 52496fig2highres.jpg &quot;/&gt; 図2： アフリカツメガエルが成熟する前と後のツメガエル卵母細胞卵成熟後、透明な白い斑点が動物半球の最上部に表示され、包細胞を剥離されている。。各アフリカツメガエル卵母細胞の直径が約1mmであるツメガエル 。 <img alt="図3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> 図3： 体外成熟卵子でから生産ICSI胚の開発、。各ステージにICSI胚の（A）の開発が要約されている。卵母細胞は、対照を注射したIVMおよびICSI続くアンチセンスオリゴヌクレオチド（対照オリゴ注入）または注射なし（非注射）、スクランブル。各段階での胚の対応する数は、バーの上に示されている。 ±SEMを示していることを意味。 N = 4-13の独立した実験/異なるFemales。（B）ICSI胚の生存例。これら尾芽段階の胚は、約200卵母細胞を出発材料として使用された一つの実験で製造した。

Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

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 ...

manipulation-vitro-maturation-xenopus-laevis-oocytes-followed

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

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

22.4K ビュー.  University of Cambridge. 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.

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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

蛍光タンパク質の発現において<em&gt; Branchiostoma lanceolatum</em&gt;未受精卵へのmRNA注入による

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

発生生物学

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

の共焦点分析を用いました<em&gt;アフリカツメガエル</em&gt; WntおよびShhのモルフォゲン勾配の調節を調査するために、

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

幹細胞様<em&gt;アフリカツメガエル</em&gt;胚外植片を早期神経発達の特徴を研究するために<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

開発にターゲットマイクロインジェクションのテクニック<em&gt;アフリカツメガエル</em&gt;腎臓

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

母親の遺伝子産物の機能操作の使用<em&gt;インビトロ</emゼブラフィッシュにおける&gt;卵成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

の胚性血管新生の可視化と定量分析<em&gt;ツメガエル</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

細胞分化中のマイトジェン活性化プロテインキナーゼ経路の光媒介可逆的調節<em&gt;アフリカツメガエル</em&gt;胚発生

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

処理と治療の男性欧州ウナギ (アンギラ) ホルモンの成熟、精子凍結保存

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

ニワトリ胚組織の開発培養法

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...