This research was supported by the Australian Research Council grant CEO348212 and the University of Newcastle. The assistance of Dr. Sam Zhang is acknowledged.

1.接合胚の開発<ol><li>植物素材<ol><li>成長のMedicagoは、14時間の光周期と23℃/ 19℃の昼/夜の温度と温室で野生型Jemalongまたはその付近の同質遺伝子（2HAとして知られている）、非常に再生成可能な遺伝子型Jemalong 2HA 13 truncatula。 <ol><li>ピアース水がシードを入力して、一晩水に浸漬させられるように種子を播種する前に（23 G針で）種皮の表面。完全種子をカバーするために十分な水を追加します。 </li><li>ポッティング混合物中の各直径15cmのポットに3種をまく（10ポットの合計）（粗砂、パーライト、コイア-泥炭（1：1：1）を加えたOsmocote正確な標準の5グラム：徐放性肥料）へと転送温室。植物が登る茎のためにポットとサラウンド当たり1植物はトレリスによってそこにあるように、発芽後の健康な苗を保持します。 </li></ol></li></ol></li><li>収穫ポッド<ol><li> Wからポッドを収集第9に記載されているように、適切な初期胚の段階を取得するためにILDタイプJemalongまたは2HA。 注：異なるポッド段は、図1に示され、対応する胚の段階は、図2に示されています。 </li><li> 表1のとおり基準を使用して、収穫時に異なるポッドステージを確認してください。別々の段階6および7（ 表2）を助けるためにステージ6からの経過時間を測定します。 </li></ol></li><li>ポッドから胚珠を解剖し、胚の段階を確認します<ol><li>必要に応じて単独で微細な鉗子を使用してポッドまたはステレオ解剖顕微鏡とピンセットから胚珠を分離します。必要に応じて胚段階を迅速に標準複合顕微鏡（ 図3B）を用いて確認することができます。 </li><li> 7.5グラムのアラビアゴム、100gの抱水クロラール、5ミリリットルグリセロール、60ミリリットルの脱イオン蒸留水を含む改変ホイヤーの溶液を調製します。 3-5時間または一晩攪拌して溶解します。 </li><li>よりリファインのためD顕微鏡は、修正ホイヤーのソリューション15,16における解剖胚珠をオフにします。慎重に透明になるまで室温で（胚珠をカバーするのに十分）ホイヤーの少量の溶液中で無傷の胚珠と場所を拾います。 </li><li>微分干渉コントラスト（DIC）光学系を有するサンプルを表示します。デジタルカメラ9（ 図2）iwth画像をキャプチャします。 </li><li>ポッドの中央領域から最も均一な胚珠を入手して、必要な数を蓄積します。例えば、 図3Aに示されています。さらに分析するために必要な温度で氷とストアの胚珠（-80°Cのための核酸）を収集します。 注：組織切片の分析の詳細については、透過型電子顕微鏡の場合、およびin situハイブリダイゼーションの論文8,9を参照してください。 </li></ol></li></ol> インビトロでの 2体細胞胚発生<ol><li> Mを使用しますtruncatula品種 T帽子は容易に培養中の胚を形成することができます。葉移植片は、このプロトコルでは2-4ヶ月の古い2HA所13,17を使用しています。 </li><li>延伸ステムの最新ほぼ完全に膨張した三つ葉の葉からチラシを取ります。 </li><li>脱水を避けるために、培養ポットに湿ったペーパータオルで三つ葉の葉をしてください。 注：三つ葉の葉が収穫されると、彼らは最小の遅延で利用する必要があります。 </li><li>培地の調製<ol><li> 9センチメートルペトリ皿で​​4：1：寒天で5培地（V 0.8％W）P4 10を使用してください。 注：P4 10：4：1：5の媒体は、P4の基礎培地18 + 10μM1-ナフタレン酢酸（NAA）、4μM6-ベンジルアミノプリン（BAP）、1μMのアブシジン酸（ABA）および5μMジベレリンで構成されてい酸（GA）。 GA + ABAの使用は、胚形成を促進し、胚の数14での実質的な増加を引き起こします。 </li><li> 1 LのP4基礎媒体を構成します。主要な塩からなる（calciuを作ります、）別途アップメートルマイナー塩およびビタミン（ 表3）、キレート鉄（別途構成する）、カザミノ酸（カゼイン加水分解物）、30gのスクロース、8グラム寒天。 </li><li>適切な分注して-20℃での主要な塩、カルシウム、カザミノ酸、マイナー塩およびビタミンの店舗在庫ソリューションを提供しています。 4℃でキレート鉄を保管してください。 </li><li> •7HのFeSO 4の1.853グラムを添加しながら攪拌しながら、脱イオン蒸留水約900ミリリットル中のNa 2 EDTA•の7.44グラム2H 2 Oを溶解させることによりキレート鉄（200X）を構成する場合、98〜99°Cの解決策をもたらします2 O.成分が溶解されたときに最後に1リットルに構成しています。 4℃で琥珀色の瓶に保管してください。 </li><li> 表4のように原液とP4の基礎媒体を構成する。培地中のホルモンは、10μMNAA、4μMBAP、1μMABA、5μMGA（ 表4および特定の材料表を参照してください）です。 NAAを追加とBAPオートクレーブ処理の前に、オートクレーブし、約55℃に冷却し、注射器に取り付けた0.22μmのフィルターで濾過滅菌を使用した後にABAとGAを追加します。穏やかな旋回によりよく混ぜます。 </li><li>オートクレーブの前に1 M KOHでpHを5.8にメディアを調整します。 20分間121℃でオートクレーブ。 </li><li>濾過滅菌ABAとGAを追加し、層流フードやバイオハザードキャビネットに無菌9センチメートルペトリ皿に約25ミリリットルのメディアを注ぐオートクレーブ後。クールで寒天は蓋をオフにして設定してみましょう。そして、蓋の上に置き、蓋には凝縮水がないことを確認してください。 （蓋コンデンセートを防ぐためにダンボール箱に）4℃で必要とストアとしてラベル。 </li></ol></li><li>葉組織の滅菌<ol><li>紫外線滅菌層流フードやバイオハザードキャビネットに働きます。 </li><li>場所は、以前にオートクレーブし、バネ茶注入器のメッシュボールに残します。 </li><li> CULエタノール：70％（V V）で葉を含む茶注入器を浸し30秒間トゥーレポット。 注：この手順のすべてのステップが使用するために250ミリリットルのオートクレーブ処理されたキャップポリカーボネート培養ポットをねじ込みます。 </li><li>ドレインその後1葉組織を転送し、浸漬：10分間希釈漂白剤（0.5％塩素）：8（V V）。随時穏やかに渦巻きます。 </li><li>漂白剤を排出し、滅菌脱イオン蒸留水を含む培養ポットに茶の注入器を転送します。優しくスワールや余分な水分を排出します。滅菌脱イオン蒸留水の新鮮な培養ポットに1より多くの時間を繰り返します。 </li><li>滅菌脱イオン蒸留水の新鮮な培養ポットに滅菌ピンセットで茶注入器から葉を削除します。無菌キャップをして反転し、旋回によってすすいでください。外植片を調製する準備ができるまで、滅菌水に浮かぶ葉のままにしておきます。 </li></ol></li><li>外植片を準備し、メッキ<ol><li>無菌技術を使用して、メスでエッジから個々の滅菌リーフレットをトリミングし、2つまたはTHRに残りの長方形をカット各外植片（ 図5A）の中心部にあるミッド静脈とEE長方形の外植片（8-10 X 3〜5ミリメートル）。 注：外植片の大きさは最初のカルスを開始で非常に重要です。持ち帰り食品容器から滅菌蓋の切削を行います。 </li><li>直径9cmのペトリ皿（ 図5B）で寒天固化培地上でプレート6の外植片。通常の葉の向きを維持するために、下に植片の背軸側にプレート。 </li><li>皿の周りに延伸パラフィルム（ 図5C）でペトリ皿を密封します。組織は現在、インキュベーションのために準備ができています。 </li></ol></li><li>組織インキュベーション<ol><li>培養期間を通して温度制御室または成長キャビネットに28℃の暗所でプレートをインキュベートします。 注：外植片は、脱分化を開始する細胞分裂、増殖（カルス形成）と胚（分化）を受けることになります。 </li><li>差別explaのサブカルチャー新鮮な培地への3週間後にNTS。この継代培養では、層流フードやバイオハザードキャビネットに、無菌のステンレス鋼製のへらと鉗子を用いて全植片を移します。カルスが発生し、カルスは、 図5Cの最大のもののサイズを超えるように新鮮な培地に継代培養したときに、個々のカルスの50％を転送します。 </li><li>約4週間の最初の胚を観察します。同期の程度はありますが、4-8週間の潜伏期間（ 図6A、B）の上に別の胚の段階を収集します。実験の目的に応じて解剖顕微鏡およびプロセスの下で収集します。 </li><li>外植片を3ヶ月間の胚を生産し続けるように、3週間ごとに継代培養し、定期的に胚を削除します。 </li></ol></li></ol>

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The authors declare that they have no competing interests.

記載されているプロトコルは比較的単純であり、すべての現代的な細胞および分子技術とマメ科植物の胚発生の調査を可能にします。我々は両方の利点と欠点は、インビボおよびインビトロの方法であることを認識しています。両方とも、未熟種子19の培養に比べて初期胚発生に関する詳細フォーカスを可能にします。 in vivo試験の場?...

収穫面積と総生産1で穀物に2つ目の豆類は、約20,000種およびマメ科（またはマメ科）家族と高等植物の第三位のファミリーです。大豆は第三位の栽培作物です。穀物豆類は、人間の消費の2のための食事性タンパク質と植物油の三分の一の約3分の1を提供します。それらのN 2の固定容量のマメ科植物はまた、持続可能な農業システムに貢献しています。 のMedicago truncatula、大豆のように、その種子の子葉にタンパク質と油を格納し、かなりの遺伝的およびゲノム資源3,4との遺伝的およびゲノムマメ科モデルです。 M.一方、 truncatulaは ​​、それがますますマメ科植物の種子生物学5-7と胚8,9を研究するために採用されているマメ科植物根粒共生4の理解の進歩を可能にしました。シロイヌナズナの胚発生は広範囲に私は10,11を研究し、それされていますSA非マメ及び胚発生の詳細はMedicagoに8,10と同じではありません。 Mの接合体胚発生truncatulaは ​​独特の多下垂、endoployploid懸垂帯および基底転送セル8と、興味深い機能を備えています。 体細胞胚形成（SE）は、一般的に、植物12を再生するために使用されます。マメ科モデルMのtruncatulaは ​​、シードラインJemalong 2HA（2HA）は体細胞胚形成13率の高さを持っている親Jemalongから開発されてきました。生産胚の数は、最近確立された実質的に長い媒体14にジベレリン酸（GA）およびアブシジン酸（ABA）の両方を添加することによって増加しています。 GAとABAは通常拮抗14を作用することが与えられた珍しい相乗的にこの場合はGAとABAの作用します。カルスから生産胚は、胚発生の段階が容易にvisuallを決定することができる表面上で開発しますY、容易に回収しました。胚形成（2HA）と非胚形成されている同質遺伝子系統（Jemalong）の近くに持つことは、体細胞胚形成の調査を容易にし、 インビボおよびインビトロ系の両方持つことは、種々の実験の可能性を提供します。 胚発生の細胞および分子メカニズムを理解することは、種子及び植物の開発を理解するために不可欠です。マメ科植物では、他の双子葉植物のように、それは、ヒトの栄養のために使用される製品を保存胚の子葉です。初期胚は、急速な細胞分裂、正しい胚のパターン形成を伴います。受精後約8日、Mのtruncatula胚は、初期の子葉段階に達します。形態学的特徴を正確に温室条件下で受精後日数で示されていません。このように、発生中の胚の段階を示すために、効率的な標準化されたアプローチは、遺伝的regulの研究に貴重なものです初期の接合体胚のエーション。 本稿では、マメ科植物のモデルMに胚発生の生物学的研究のために開発した胚を収集するために、2つの標準化されたプロトコルを提供しますtruncatula。第1は、第二は、簡単にアクセス大きな胚番号を提供するために、培養葉移植片を介して、体細胞胚形成の間、胚発生およびポッド形態を関連付けることにより、接合胚を収集することです。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >P4 medium</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Use Sigma-Aldrich Chemicals or other analytical grade supplier</td>
		</tr>
		<tr >
			<td >Major salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Minor salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vitamins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Bacto Laboratories</td>
			<td>214010</td>
			<td>Bacto agar</td>
		</tr>
		<tr >
			<td >Plant hormones</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1-Naphthaleneacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0640</td>
			<td>Dissolve in small amount of 1 M NaOH</td>
		</tr>
		<tr >
			<td >Abscisic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A1049</td>
			<td>Dissolve in small amount of 1 M NaOH</td>
		</tr>
		<tr >
			<td >6-Benzylaminopurine</td>
			<td>Sigma-Aldrich</td>
			<td>B3274</td>
			<td>Dissolve in MQ water with heating and few drops 1N HCl</td>
		</tr>
		<tr >
			<td >Gibberellic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>G7645</td>
			<td>Dissolve in small amount of ethanol</td>
		</tr>
		<tr >
			<td >Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Stereo dissecting microscope</td>
			<td>Leica</td>
			<td>MZFLIII</td>
			<td>Or similar</td>
		</tr>
		<tr >
			<td >Light microscope</td>
			<td>Zeiss</td>
			<td>Axiophot</td>
			<td>Or similar, with suitable optics</td>
		</tr>
		<tr >
			<td >Digital camera</td>
			<td>Zeiss</td>
			<td>AxioCam HRc</td>
			<td>Or similar</td>
		</tr>
		<tr >
			<td >Sterilising leaves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >250 mL screw cap polycarbonate container with polypropylene lid</td>
			<td>SARSTEDT</td>
			<td>75.9922.519</td>
			<td>Autoclavable</td>
		</tr>
	</tbody>
</table>

protocols for obtaining zygotic and somatic embryos for studying the regulation of early embryo development in the model legume medicago truncatula

単一細胞受精卵から始まる初期胚は、急速な細胞分裂や形態形成を通過し、形態学的に前球状、球状、心臓、魚雷と子葉の段階によって特徴づけられます。この進歩的な開発は複雑な分子ネットワークの厳しい規制下にあります。開発の同様の段階で十分な初期胚を収穫することは初期胚の細胞および分子の規制を調査するために不可欠です。 Medicagoにするための、例えば 8日早い子葉段階に到達するためにtruncatulaながら初期胚発生が短い急速な形態形成を受けるので、これは簡単ではありません。ここでは、二つのアプローチによって問題を解決します。最初のものは、接合胚の段階を示すうえで胚発生とポッドの形態との間の結合を確立します。これは、特にポッドスパイラルと棘の開発の数に基づいています。 in vivoでのを補完する別の方法tudiesは体細胞胚を生産するために培養葉の外植片を介してです。オーキシン（1-ナフタレン酢酸）、サイトカイニン（6-ベンジルアミノプリン）、アブシジン酸およびジベレリン酸 - 媒体は珍しいホルモンの組み合わせを含みます。異なる段階は、解剖せずに、カルスから成長している識別することができます。

F -異なる胚の段階は、 図2Aに示されているがF -接合体胚形成のために異なる胚段階に対応する異なるポッド構造が図1Aに示されています。同じ段階でポッドを選択することにより、非常に均一である胚珠のサンプルは、（ 図3A）を得ることができます。 RT-qPCRの胚を使用して、特定の遺伝子が容易に検出され、経時的研究は?...

Watch this Scientific Journal Video about Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula at JoVE.co

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

モデルマメに初期胚発生の調節を研究するための接合体と体細胞胚を得るためのプロトコル<em&gt;のMedicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

protocols-for-obtaining-zygotic-somatic-embryos-for-studying

Research

JoVE Journal

Developmental Biology

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

13.8K ビュー.  University of Newcastle, Callaghan, New South Wales, Australia. The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided. 

ビデオ: Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

エバンスブルー色素でゼブラフィッシュ幼生骨格筋の整合性の分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

発生生物学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

リビングゼブラフィッシュ胚における細胞内構造のイメージング

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

のトランスクリプトームプロファイリング<em&gt;生体内で</em二色マイクロアレイプラットフォームを使用&gt;産ウシ着床前の胚

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

ゼブラフィッシュ初期胚および腫瘍の細胞の電気的活動の可視化

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

ウサギモデルにおける最適胚期における低侵襲胚移植と胚ガラス化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

初期マウス胚からのシーケンシングのための小型RNAライブラリーの調製

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

初期ショウジョウバエ胚における免疫組織化学およびRNAのその場分布のためのプロトコル

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

ゼブラフィッシュ胚の簡易かつ効果的な移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

インスリン産生細胞への分化のためのラット脂肪組織間葉系幹細胞の単離

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

エンフェイス マウス胚の平面形態形成解析のための心内膜クッション作製

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...