アルバータ州畜産食肉エージェンシー株式会社、豚肉CRC、アルバータ州の豚肉、Hypor Aヘンドリックス遺伝当社及びNSERC CRSNG：著者は協力し、以下の研究助成機関の財政的貢献を感謝したいです。

動物実験のガイドラインにカナダの評議会によると、および学部アニマル政策と福祉委員会の承認を得て - ア​​ルバータ大学の家畜、妊娠中の雌豚は、妊娠と胚の30を回収した約デーで訓練を受けたスタッフにより安楽死させました。手順の間にすべての回で検査用手袋を使用してください。 1.サンプル収集およびサンプル保管<ol><li>放血に続いてキャプティブボルトを使用して雌豚を安楽死させます。各安楽死させた雌ブタから生殖器および胚を収集するために、検査用手袋を着用してください。 注：氷や液体窒素を乾燥させるサンプルと移植胚の急速なコレクションは、マイクロアレイや定量PCRなどの他の分子作品のための高品質の組織になります。 </li><li>子宮を解剖し、静かに、基礎となる子宮壁21から自分の胚体外胎盤膜内のすべての胚を分離します。何の母体組織の汚染がないことを確認してください。 </li><li>リ凍結前に、すべての実行可能な胚のコードの長さと重さ。 </li><li>各個々の胚を収集し、すぐにスナップを液体窒素で凍結する前に、アルミホイルでそれを包みます。 </li><li>直接-80℃の冷凍庫に液体窒素から凍結した胚を移します。 </li></ol> 2.研削胚<ol><li>分析されるサンプルの数に必要な検体IDと全てのサンプル試験管（15ml）にラベル。 </li><li>半分にドライアイスと熱的にコンテナを記入し、ドライアイスで予め標識試料管を挿入します。 </li><li>ドライアイスの容器に℃の冷凍庫-80からあらかじめ冷却モルタルおよび乳棒を転送するために上に検査用手袋の別のペアで冬の手袋を着用してください。 </li><li>ドライアイス上のモルタルの内側に凍結した胚を置きます。 </li><li>胚をカバーし、微粉末に乳棒でそれを粉砕するのに十分な液体窒素を注ぎます。 </li><li> MICRで予め標識したサンプル管に胚の粉末を移しますospatula（ 図1）し、-80℃の冷凍庫にチューブを配置します。 </li><li>各胚のためのクリーンあらかじめ冷却し、乳鉢と乳棒を使用して、サンプル間の交差汚染を避けるために、各胚を研削した後、検査用手袋を変更します。 </li></ol>変更された水酸化ナトリウム法を用い3.ゲノムDNAの準備<ol><li>サンプルの数のために必要なラベル全て微小遠心管（1.5ml）を分析します。 </li><li>前の出発にドライアイスの入った容器にから胚粉末を含むサンプルチューブ-80℃の冷凍庫を転送します。 </li><li>各前標識されたマイクロ遠心チューブに50mMのNaOHで（水酸化ナトリウム）の180μLをピペット。 </li><li> 95℃までインキュベーターと予熱をオンにします。 </li><li> 50mMのNaOH溶液を含む前標識されたマイクロチューブにサンプル管から胚粉末の正確な量（約5〜10 mg）を（ 図2）を転送するためにつまようじを使用してください。 </li><li> Uを引いゆっくりと粘着性、ねばねば、白い透明状物質（ 図3）のようにDNA溶解液を可視化するPと同じつまようじ。 </li><li> 5分間95℃で予備加熱したインキュベーターにDNA溶解液でマイクロ遠心チューブを転送します。直ちに氷で満たされた発泡スチロールの箱にチューブを移します。 </li><li>直接マイクロ遠心チューブに1 Mトリス塩酸の20μLを加え、穏やかにチューブをタップして、それを混ぜます。 pHは遠心分離前に約8.0（ 図4）であることを確認するためにpH試験紙を使用してください。 注：サンプル調製の多数を扱う場合には、ランダムにpH値を確認するために、いくつかのサンプルを選択するために許容可能です。 </li><li>未溶解組織片を除去するために、室温で2分間微量で2000×gでDNA溶解液でチューブを遠心。 </li><li>新しいチューブまたは96ウェルプレートにトップ透明な上澄み150μlのを転送します。 注意：明確なDNA溶解液は、PC内のテンプレートとして使用する準備ができていますRの反応、それは2週間、4℃で安定である、またはそれは年間-20℃で保存することができます。 </li></ol> 4.デザインセックス特異的PCRプライマー<ol><li> NCBIウェブサイトhttp://www.ncbi.nlm.nih.govからブタ性別決定領域Y（SRY）のためのアクセッション番号（NM_214452.3）と、亜鉛フィンガータンパク質のX連鎖（ZFX）遺伝子（XM_005673501.1）を取得/。 </li><li>コピーし、オンラインプライマー設計ツールにこれらのNCBIアクセッション番号を貼り付けます。 注：長さを含む最高のプライマー情報、TmとGC％だけでなく、PCR産物のサイズ（bp）が、コンピュータの画面（ 図5）で生成されます。 </li><li>これらの配列のみをそれぞれSRYとZFX（ 図6）のために、XとY染色体上に位置していることを確認するために、現在のブタゲノムデータベース104に対してヌクレオチドBLASTプログラム（BLASTN）を使用して、これらのプライマーの特異性を検証します。 </li></ol>直接DNA溶解物から5.ゲノムDNAをPCR <ol><li>使用15μlのPCR反応の鋳型としてDNAの溶解物のみ1μlの。 注：96ウェルプレートPCRのためのハイスループットスクリーニング方法は、マルチチャンネルピペットを用いて同様に行うことができます。 </li><li>ちょうど最初のPCRのために次の操作を行います。商業的に入手知らセックスブタのゲノムDNAの1μL（0.5 ngの）を追加することにより、鋳型なしの陰性対照および陽性対照用の2つの追加のチューブのための1つのPCRチューブを準備します。その後、ポジティブコントロールとして最後に成功した雌雄鑑別分析からサンプルを含み、セックス決意のための新たなPCR反応と一緒に実行されます。 </li><li>任意のHotStartの準備ができてミックスPCR酵素を使用して、PCR反応の合計数に応じてプライマーおよびヌクレアーゼフリー水を添加することにより、マスターミックスを準備します。 PCRマスターミックスの14μlの既存のPCRチューブにDNA溶解液を1μlを追加します。 2性別特異的遺伝子からプライマーの最終濃度は15μlのPCR Rの合計で0.3μMであるようにプライマーを追加します。eaction。 </li><li>サーマルサイクラーで、以下のPCRプログラムをセットアップします：3分、95℃35サイクル15秒の伸長工程に続いて65℃で15秒のアニーリングステップに続いて20秒の溶融工程98°C、各72℃。最後のステップにおいて、72℃で1分間、反応をインキュベートし、性別を決定するために、ゲル電気泳動の検証のためのPCRチューブを除去するまで4℃でインキュベートし続けます。 注：PCR条件および最適化は、 材料表に記載されたPCRキットのマニュアルに従ってあります。 </li><li> 2％TBEアガロースゲルを調製する場合、非毒性緑蛍光SYBR DNAゲル染色の適切な量を追加します。 注：それは利用できない場合エチジウムブロマイドは、緑色蛍光染色のために置換することができます。 </li><li> PCRチューブにローディング色素（10倍）の1.5μlを添加して、PCR反応緩衝液アップダウンをピペッティングしてよく混ぜます。 </li><li>ウェルおよびRへの試料の負荷を10μl（染料バンドがゲルを通って途中で実行されるまで、小さなゲル装置100 V、150 Vで96ウェルのゲル装置で）適切な電圧設定で未アガロースゲル。 </li><li>観察し、レーザースキャナを用いて蛍光灯の設定の下でバンド強度を調整し、ゲルの画像を取り込みます。注意：一般的なゲルイメージャは、エチジウムブロマイドゲルの代わりに用いることができます。 </li><li>女性としての一つのバンドと男性のような2つのバンド（ 図7）で胚を識別することにより、ブタ胚の性別を決定します。 </li></ol>

<ol>
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著者は、彼らが競合する金融利害関係を持たないことを宣言します。

ブタ胚のDNAセックスタイピングに関連する既存のプロトコルのほとんどは早期着床前段階15,16に適しています。我々が正常に妊娠後期の間、ブタ胚のスクリーニングに適したプロトコルを開発しました。以前の研究6,18からの胚の同様の発達段階での研究に基づき、本プロトコルは、安全で低コストであると考えられています。 このプロトコルは、96ウェ?...

家畜ブタはヒトおよび家畜の分野での開発、遺伝学や栄養学の基本的な研究テーマとなっています。人間の研究のための生物医学モデルとして豚の電位は、それらの生理学的類似性に起因することができます。家畜では、性比の操作は、選択と遺伝的改良プログラム1の有効性を高めることができます。個々の胚を雌雄鑑別することは、遺伝子型、エピジェネティクスと初期胚の発育2時の性的二型のX不活性化を含むがこれらに限定されない多くの実験的調査に使用される基本的なツールです。 マウスでの研究は、母体の食事やその他の要因は男女の不均衡3をもたらし得ることを示唆しています。豚では、男女比の不均衡の原因は父方の品種4、子宮容量5と、母豚の代謝条件6が含まれます 。胚および同腹仔で観察された差異は、それ自体によって影響され得るのでxual二形は、研究者が自分の研究に関する結論を描画する前に胚のセックスと性比に注意する必要があります。開発の30日目にブタの胚を雌雄鑑別するための効率的なツールやプロトコルの開発は、ここで議論されます。 セックスタイピングの様々な方法がモデル生物と家畜の遺伝的研究のために開発されてきました。特に家畜で、男性と女性の初期胚を識別することは繁殖プログラムのための遺伝子選択性を高めるために非常に一般的です。ギムザ7または8,9の技術はセックスタイピングのために使用されている強い蛍光を使用して、ブタにおける初期胚の染色体分析。しかしながら、これらの方法は時間がかかり、迅速かつ正確に胚の多数をスクリーニングするには適していません。 最も効果的な性別のタイピング方法は、熱安定性DNAポリメラーゼおよびプライマーの対を用いてDNAの増幅です。 PCR法によるDNAの雌雄鑑別は、o-、より、具体的な迅速かつ高感度でありますNLY細胞物質の微量を必要とします。最初のPCRベースの胚性判別は、ヒト10、およびそれ以降のマウスでは11、牛12、水牛13と羊14着床前胚で実施しました。豚では、最古のDNAのセックスタイピング法は、Y染色体特異的なDNAプライマー15の単一の対を介して着床前胚のために設立されました。しかし、性別決意のための最も一般的なPCRプライマーは、雄特異的なSRY遺伝子16とXとY染色体17の両方に位置するジンクフィンガー遺伝子の非性別判別領域のY染色体から選択しました。その後、これらのプライマーは、ジンクフィンガー遺伝子の唯一のX-染色体を検出するためのプライマーの改善された特異性を持つこの研究で30日目の胚の性別を決定するために適用されてきました。 ブタ着床前胚からのゲノムDNAをproteinasでバッファする無傷の胚盤胞を露出することによって抽出することができますE K 16または15個々の初期卵割胚から少数の細胞の生検を取り、直接PCRのためにそれらを使用します。しかし、大きさが数センチメートル以上ブタ血液、毛髪、組織または大受胎からのDNAの放出を効果的にプロテイナーゼK法を使用して行われていません。これらの材料のためのDNAの抽出方法は、時間がかかり、フェノール/クロロホルムプロトコル6または高価な列ベースのキット18のいずれかを使用して確立されています。潜在的に有害な化学物質の使用を回避するために、安価で、簡単で、フェノールを含まないDNA抽出方法を開発する傾向があります。マウス19およびゼブラフィッシュ20組織からのPCR品質のゲノムDNAを単離するためのプロトコルのこのタイプのホット水酸化ナトリウム及びトリス（ホットショット）を使用して確立されています。本研究では、変更された名手でDNAを得るためのプロトコルを提供し、日の細胞溶解物から直接で30ブタ胚を入力してPCRのセックス二重のプライマー対を再設計しました高精度。

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">KAPA HiFi HotStart Ready Mix PCR kit&#160;</td>
			<td style="width:203px;">KAPABiosystems</td>
			<td style="width:189px;">KR0370</td>
			<td style="width:463px;">other Hot Start Taq polymerase can be used after optimization</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:189px;">S33102</td>
			<td>Ethidium bromide can be substituted for SYBR Safe</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Pig female and male genomic DNA</td>
			<td style="width:203px;">Zyagen</td>
			<td style="width:189px;">GP-160-F1 &#38; GP-160-M5</td>
			<td>Postive controls from the tissues of known sex DNA can be used.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Typhoon FLA 9500 laser scanner</td>
			<td style="width:203px;">GE Healthcare Life Sciences</td>
			<td style="width:189px;">28-9969-43</td>
			<td>other imaging system can be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Free Soft Nitrile Examination Gloves</td>
			<td style="width:203px;">WWR</td>
			<td>89038-270</td>
			<td>any other examination glove can be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Sodium hydroxide, solid&#160;</td>
			<td style="width:203px;">Fisher</td>
			<td style="width:189px;">BP 359 -212</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Eppendorff DNA LoBind Tubes, 1.5 ml, PCR clean</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">0030 108.051</td>
			<td>heat resistant</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">ThermoStat plus</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">22670204</td>
			<td>Use as a incubator for 95C, don&#39;t need to use the heater</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Toothpick</td>
			<td>Bunzl Plc</td>
			<td style="width:189px;">75200815</td>
			<td style="width:463px;">Any round wooden toothpicks can be used - quality wood</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Microcentrifuge 5417R/5417C</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">22621807</td>
			<td>This model was discontinued. But another newer model can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microspatula</td>
			<td style="width:203px;">Fisher</td>
			<td style="width:189px;">SDI28540115</td>
			<td>Autoclaved before use each time.</td>
		</tr>
	</tbody>
</table>

accurate and phenol free dna sexing of day 30 porcine embryos by pcr

豚における出生前のプログラミングの研究は、胚または胎児の性別が発達アウトカムに影響を与えることができることを示しています。したがって、胚の性別を決定する能力は、特に初期の開発に関する多くの実験に必要です。本プロトコルは、PCRで使用するための豚ゲノムDNAの、安価で迅速かつ非毒性の製造を示します。 30日目の胚は、人道的に存在するプロトコルのために施設内動物政策と福祉委員会によって確立されたガイドラインに従って収集されなければなりません。このPCRベースの雌雄鑑別技術のための全胚の作成は、単純にあらかじめ冷却し、乳鉢と乳棒を用いて微細な粉末に凍結した胚を粉砕することを含みます。 PCR品質のDNAをアルカリ溶解試薬ホットインキュベーションを適用することによって、胚の粉末の少量から放出されます。次に、DNA溶液を中和緩衝液と混合し、PCRに直接使用しました。二つのプライマー対はdetecに生成されます高精度と特異性を有するX-染色体のY-染色体（SRY）とZFX領域の領域を決定トン特定セックス。同じプロトコルをまた、30日目より前の他の細長い胚（14日目まで10日目）に適用することができ、胚の多数をスクリーニングする自動化が可能作り、高場合、このプロトコルは、96 welledプレートで実施することができますスループットセックスタイピング。

PCRによって345 DNA溶解物スクリーニングから性別決意の代表的結果を図7 に示し、表1に要約します。 図7に見られるように、65℃でのプライマーアニーリング温度が異なるサンプル間で同様の強度及び述語アンプリコンサイズ（ 図5）を生成する ?...

Watch this Scientific Journal Video about Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR at JoVE.com

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

PCRによる日30ブタ胚の正確かつフェノールフリーDNA雌雄鑑別

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

accurate-and-phenol-free-dna-sexing-of-day-30-porcine-embryos-by-pcr

Research

JoVE Journal

Developmental Biology

9.9K ビュー.  University of Alberta. This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

ビデオ: Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

クロマチンレギュレータと米国でのゲノムワイドスナップショット<em&gt;アフリカツメガエル</emChIPの-配列によって&gt;胚

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

発生生物学

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

胚の蛍光標識でDNA特異的染色メチルグリーンの応用

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

早期着床後のマウス胚への細胞やDNAの正確​​ローカライズ転送する方法

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

ゼブラフィッシュ胚からの単一細胞の単離とキャラクタリゼーション

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

胞胚の開発の外科的融合による並体結合ゼブラフィッシュ胚の生成

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

生きた蛙およびゼブラフィッシュの胚における低酸素の誘導

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

ラミニン521マトリックスを用いたヒト誘導多能性幹細胞の統合された誘導

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

心血管イメージング (30/45 MHZ) の高周波超音波を用いた胎児マウス

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

キセノプス・レービス胚における眼芽へのDNAのマイクロインジェクションとGFPのイメージングは、無傷で光学軸軸樹園を発現し、生きているキセノプス・タマジャクシ

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

ゼブラフィッシュ胚と幼虫の全山免疫賦形化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...