This work was supported by grants from the Deutsche Forschungsgemeinschaft to SB (BR-2215; SFB 497/A9).

注：全ての動物実験は、ドイツの法律に基づいて実施し、テュービンゲンの政府のオフィスによって承認された。 マイクロピペット、ソリューションおよび膜の作製<ol><li>マイクロピペットの作製<ol><li>プル、540：125、ベロシティを：熱：以下のプログラムでマイクロピペットプラーを用いてガラスマイクロピペットを引いて20とディレイ：140針の長さは5.5センチメートルにのぼる。 </li><li> microgrinderを用いてベベル針は4mmの適切なチップサイズを取得する。先端の損傷を防ぐために、ボックス内の針または15センチメートルペトリ皿に保管してください。 </li></ol></li><li>溶液の調製<ol><li>プラスミドDNAソリューション<ol><li>製造業者のプロトコルに従ってエンドトキシンフリーマキシプレップキットを用いて、所望のcDNA構築物を含むプラスミドDNAを調製する。 </li><li> （GFPスパイクベクトルなし）/μL3または4μg/最終濃度をプラスミドDNA溶液を調整しますファストグリーン（最終濃度0.05％）を含有するエンドトキシンフリーのTris-EDTA緩衝液中で（GFPスパイクベクター1μgを有する）ずつ。 </li></ol></li><li>ラミニン原液<ol><li>最終容量1mlに滅菌水でラミニン1mgを溶解させる。 -80℃で100μlのアリコートとストアを準備します。 </li></ol></li><li>ポリ-L-リシンストック溶液<ol><li> 1mg / mlの最終濃度に滅菌水50ml中のポリ-L-リジン50mgを溶解する。 -20℃で1ミリリットルアリコートとストアを準備します。 </li></ol></li><li>ハンクス液を完了します（完全HBSS） <ol><li>のCaCl 2を10mlの100mMの硫酸マグネシウムを 10×HBSS 100mlを、1 M HEPES緩衝液を2.5ml（pH7.4）中、1 M D-グルコースを30ml、100ミリモル、10mlのを組み合わせることにより、完全なHBSSを作成し、4 1 mlのMのNaHCO 3。 4℃で1 Lとストアに滅菌水を上に追加します。 NOTE：フィルであるオートクレーブ全ての溶液を1M HEPES緩衝液、1MのD-グルコースを期待TERは、滅菌した。 </li></ol></li><li>スライス培養培地<ol><li>基礎培地イーグル35mlの完全HBSS（1.2.4）12.9 mlの1 M D-グルコース、200mMのL-グルタミンを250μl、およびペニシリン - ストレプトマイシンを500μlの1.35を加えて、スライス培養培地を調製50ミリリットルの最終容量を得る。 4℃で最終濃度5％とストアにウマ血清を加える。 </li></ol></li><li>低融点（LMP）アガロース<ol><li>ハイパワーで1〜2分間電子レンジで加熱して完全なHBSS（1.2.4）の50ミリリットルにアガロースLMPの2グラムを追加することで、4％LMPアガロース溶液を準備します。 39℃ - 37℃の水浴中でこのソリューションを保管してください。 4℃と再利用で溶液を保管してください。 </li></ol></li><li>パラホルムアルデヒド溶液<ol><li>ドラフト内で1×PBS 100mlにパラホルムアルデヒドを4g添加することにより、4％パラホルムアルデヒド（PFA）溶液を調製する。 60°Cまで溶液を加熱し、溶液をcになるまで1 NのNaOHを数滴加えるリア。 </li></ol></li><li>透過処理ソリューション<ol><li> 4℃で0.3％Trition X-100およびストアを含む1×PBS 300mlにBSAを9gに溶解する。長期保存のために10％のアジ化ナトリウムを加える。 </li></ol></li></ol></li><li>メンブレンインサートのコーティング<ol><li>ラミニンストック溶液（1.2.2）の一方のアリコートおよび12 ​​mlの最終容積に滅菌水でポリ-L-リシンストック溶液（1.2.3）のいずれかのアリコートを希釈する。 </li><li>滅菌水を含む各ウェル2mlで6ウェルプレートに膜インサートを配置します。膜の上にコーティング液1 mLを加え、5％CO 2インキュベーター中、37℃でO / Nインキュベートする。 </li><li>インキュベーション後、膜が滅菌水とドライの1ミリリットルで三回を挿入洗う。ドライ6ウェルプレートに4週間まで4℃で同日またはストア上にコーティング膜インサートを使用してください。 </li></ol></li></ol> 2. DNA注入及びE15.5のエレクトロポレーションおよびE18.5胚<ol><li&gt; 5％イソフルランで飽和させ、気化器に接続された麻酔ケースにそれを置くことによって、雌マウスを交配時間を麻酔。 1 L /分の速度でイソフルランと酸素を循環させる。マウスの足の間でつまんでテストされて2-4分間または無意識になるまでボックスに動物を保管してください。 </li><li>胚日（E）15.5または18.5に頸椎脱臼によって無意識のマウスを安楽死させる。胚13を含む子宮を解剖し、冷たい完全なHBSS 15〜20 mlを含有するペトリ皿に入れてください。 注：この時点以降から、氷上で胚組織を保つ。 </li><li>冷たい完全なHBSSを含む第二のペトリ皿に子宮角と場所から各胚を分離するためにハサミを使用してください。 </li><li>解剖顕微鏡下では、子宮筋壁と細かい鉗子（＃55）とハサミを使用して胎盤を断つ。慎重に卵黄嚢から胚をリリース。 </li><li> decapitatするボンハサミを使用してくださいちょうど60°の角度で前肢上記電子胚。実験は、胚の遺伝子型決定を必要とする場合、ゲノムDNAの単離のための組織試料（尾の小片）を収集する。 </li><li>清潔で乾燥したペトリ皿に頭を転送します。ヘッドは60°の角度で断頭されていたので、背側を上に置かれたとき、頭が片側に傾けなければならない。 </li><li>ブレグマに半球近いの中央に慎重に針を置きます（ 図1A、B）。 5-8パルスを印加する、パルスあたり10〜15ミリ秒の間、圧力の30ポンドを使用してpicospritzer IIIのペダルを踏んでDNA溶液の（3または4μgの/μLで）約2〜3μLを注入。パルスの持続時間及び数は、より多くの時間を必要とする小さな開口部を有する針開口部の直径に依存する。各パルスの間隔は1秒に達する。 </li><li>電極を配置する前に、EMBRの頭の上に完全なHBSSの数滴を適用ヨーヨー。 「負」の端末が注入心室および胚の頭部（ 図1C、D）の耳の下に注入された心室の反対側に「正」電極と同じ側になるように電極を配置する。 50 Vの5パルスを適用します<ol><li> E 18.5のためのE 15.5と5ミリメートル電極用の3mmの電極を使用してください。 </li></ol></li></ol>脳の3.解剖<ol><li>エレクトロポレーション後、細かいピンセットの助けを借りて、頭から皮膚をはがし。春のハサミを使用すると、頭蓋骨の正中線での小脳の真ん中に小さな切開を作る。 </li><li>切開部に春のはさみを挿入し、矢状縫合に沿って縦に切断。頭蓋骨をはがし、微細な鉗子を使用して頭蓋骨から脳を切り離す。 15〜20ミリリットル冷たい完全なHBSS溶液中に脳全体を転送します。 </li><li>一方、4％LMPアガロースを注ぐ剥ぎ取り金型に、水浴中で37〜39℃に保った。 </li><li>小さなスクープスパチュラで完全なHBSSの中から脳を取り、細かいティッシュペーパーやキムワイプを使用してHBSSの過剰を排出。 </li><li>アガロースに優しく全脳を置き、細い針との位置を調整します。アガロースが固化され、ブロックは（冠状切片のために嗅球はポイントアップ）区分されるまで氷上に金型を保管してください。 </li></ol> 4.ビブラトームセクショニングとスライス文化<ol><li> LMPアガロースブ​​ロックをトリミングし、「スーパーグルー」を使用して試料ステージにそれらを接着。ブロックが溶液に浸漬されるまでHBSSを完了コールドグルーの後ビブラトームのバッファトレイに試料ステージ乾式転写され、氷で埋める。 注：セクショニング前に70％エタノールですべての楽器や機器の表面を滅菌する。 </li><li>続いて、新しいブレードを使用して250μmの厚さのビブラトーム切片を準備します。 <ol><li>ブロックのWiトリム以下の設定番目。周波数 - 60Hzの、振幅 - 0.7μmで、速度16〜18ミリメートル/秒。低速（9ミリメートル/秒）での上記の設定で所望の組織を含むセクションをカット。後脳から切片を開始し、大脳から5-7のセクションを集める。 </li><li>曲がったへらの助けを借りてHBSSを完了し、すべてのセクションが（ 図1E）を収集するまで氷上に保つの氷冷5ミリリットルを含むクリーンな6ウェル培養皿にセクションを転送します。 </li></ol></li><li>セクションの向きを容易にするために、膜上のセクションを配置する前に完全なHBSS100μlのクロスファッションで膜を湿らせます。 </li><li> （鉗子でセクションのコーナーをピックアップし、へらの上に引っ張ると、膜上にセクションをプッシュする鉗子を使用してください）​​メンブレン上に曲がったへらを使用してセクションを転送します。 1膜上の5つのセクションにまで配置し、鉗子を使用して手配します（ 図1F）。互いに部分を重複しない。 <ol><li>ピペットを用いて膜からHBSSの過剰を取る。ここで使用される特定の膜は、フレームに取り付けられ、組織が接触することを可能にするが、媒体によって覆われていない組織培養プレートに挿入される。 </li></ol></li><li>スライス培養培地の1.8ミリリットル（1.2.5）（ 図1G、H）を含む6ウェルプレートに膜インサートを配置します。 11 DIVまたは14 DIV 5％CO 2、37℃で培養皿をインキュベートする。半分培地（0.9ミリリットル）ごとに二日を変更します。 注：;標識は培養時間の最初の20時間のメディアに増殖している細胞については、この段階では、ブロモデオキシウリジン（10μM最終濃度のBrdU）のような試薬を追加します。 </li></ol>免疫蛍光染色したセクションの5.固定<ol><li>の向きに応じて膜をカットしてトリミングするクリーンでシャープなメスの刃を使用して、セクション。 </li><li> 4％PFA（1.2.7）の1ミリリットルを含む24ウェルプレートに膜と一緒にセクションを転送します。 15分ごとに、1×PBSで3回洗浄し、室温で1時間のセクションをインキュベートします。静かに撹拌しながら4℃で透過処理溶液でのセクションのO / Nをインキュベートする。 </li><li>翌日、透過処理溶液中に希釈された適切な一次抗体と共に切片をインキュベートし、O / Nまたは穏やかに撹拌しながら4℃で48時間。 </li><li> 1×PBSで15分間、切片を3回洗浄し、透過処理溶液中に希釈された適切な二次抗体と共に4℃でO / Nインキュベートする。 </li><li>二次抗体とのインキュベーション後、10分間DAPI染色に続いて15分間1×PBSで一回のセクションを洗う。 </li><li>セクション15分間1X PBSで3回ずつ洗浄し、スライドを顕微鏡に移す。 ImmunoMountを加え、穏やかにセクションの上にカバースリップを配置。 4℃、それ自体でスライドO / Nを乾かしマニキュアとアル。 注：常に4℃でスライドしてください。 <ol><li>共焦点顕微鏡（ 図1I）でスライス培養を分析します。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

海馬は、学習および記憶において重要な機能を有している。歯状回も神経発生は、開発中だけでなく、成人期を通じてだけでなく、発生した2脳領域の一つです。産後と多くの共通の要因が関与する類似の方法で、大人の海馬の神経新生が進行する。これらの要因の調節機構を定義すると、今度は新たな治療法や予防措置につながる神経変性疾患を理解する上で非常に参考になります。この情...

海馬は記憶や学習に重要な役割だけでなく、感情的な行動を果たしている。一つの主な機能は、神経系の高い可塑性を必要とする長期記憶に短期記憶の統合、から構成されている。海馬の歯状回は、入力された情報のための主要なゲートウェイとして機能し、また成人期1,2を通じて継続的なニューロン新生と2脳領域の一つです。海馬の構造の開発が遅れて胚形成の間、特に最初の3〜4週間の生後3中に発生します。歯の早期開発中の幹細胞プールは、出生後だけでなく、大人の神経発生4に必要な確立されている回。現像ニューロンは、生後ならびに成体神経発生の間に、未成熟し、最終的に成熟したニューロンの前駆細胞のいくつかの段階を経て幹細胞から、様々なステージを通過する。神経発生の異なる段階での発現特定の遺伝子が海馬の回路5,6に新しい神経細胞の成熟と統合を可能にするために必要とされている。 免疫組織化学により、マウス遺伝学および表現型分析を用いて、ならびに分子的方法は、これらの遺伝子の多くの発現パターンおよび機能を定義することができた。さらにマイクロアレイ分析、ならびにクロマチン免疫沈降法（ChIP）の潜在的な直接的および間接的な標的遺伝子7,8についての情報を提供した。しかし、歯状回の特定の開発における海馬開発の調節機構に関する多くの未解決の問題は、まだあります。さらなる洞察を得るためにダウンや、興味のある遺伝子及び/又はその標的遺伝子のアップレギュレーションおよび開発中に、それらの運命を追跡する少数の細胞の操作を可能にする必要とされるどの特定の遺伝子系を規制されている。 子宮内エレクトロポレーションで shRNAは、目的の遺伝子のcDNAまたはCreリコンビナーゼのrecombinaSEはそのようなツールを提供しています。確実にするために、所望のDNAまたは低分子RNAの発現プラスミドの存在は、エレクトロポレーションのために使用されるべきである。このアプローチは非常に成功した皮質の開発9,10の勉強に実装されますが、より深い脳の層における海馬の構造の位置に起因する歯状回の開発を検討し、より挑戦的なアプローチです。 器官スライス培養に続いて子宮外エレクトロポレーションは、この問題11,12を回避する一つの方法です。 子宮内エレクトロポレーションではない全胚とは対照的にのみヘッドしたがって、海馬および歯状回に向けたshRNA / DNAを指示するためのより有利な方法で電極を配置することを可能に用いられる。私たちのグループは正常に歯状回の開発8時の転写因子Bcl11bの役割を研究するために子宮外エレクトロポレーションを採用。 Bcl11bは、rによる歯状回開発の二重の役割を持っている免疫組織化学によって実証されたように、前駆細胞の増殖ならびに分化をegulating。さらに、これらのプロセスにおけるBcl11bの関与のためのメカニズムを定義するには、Polleuxグループ11,12のプロトコルは、プロトコルセクションに下記のように歯状回を研究するために調整した。最初のアプローチでの質問はBcl11bが自律神経細胞分化細胞を規制されているかどうかに対処した。第二のアプローチは、デスモプラキン、Bcl11bの直接の標的遺伝子は、Bcl11b表現型を救出するのに十分であるかどうかを検討した。

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ex utero electroporation and organotypic slice culture of mouse hippocampal tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

転写因子Bcl11bのアブレーションは、前駆細胞の増殖および減少歯状回のサイズと細胞数を生じる神経分化の障害を引き起こす。さらに、変異体ニューロンは、学習と記憶障害8を引き起こして海馬回路に統合することができない。 子宮外これらのプロセスでBcl11bの調節機構（複数可）に関する質問に答えるためにエレクトロポレーションを用いた。 <p class="jove_content"...

Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

子宮外エレクトロポレーションおよびマウスの海馬組織の器官型スライス培養

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

ex-utero-electroporation-organotypic-slice-culture-mouse-hippocampal

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

9.4K ビュー.  University of Ulm. Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

ビデオ: Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

タンパク質の削除の誘導を介して<em&gt;子宮内で</em&gt;エレクトロポレーションは、トランスジェニックモデルにおいて神経細胞の移動の欠損を定義する

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

発生生物学

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

初代細胞培養研究のためのひよこ網膜における効率的な遺伝子導入：<em&gt;元オボ</em&gt;エレクトロポレーションアプローチ

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

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 ...

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

生後神経発生の研究のための器官型スライス培養

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化と共培養マウスの卵巣と卵胞

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

チック小脳スライスと顆粒細胞前駆体の空間的にターゲットを絞ったエレクトロポレーションの ex vivo培養

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

郭清とコロナのスライス標本マウス下垂体腺の開発

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR を介した損失関数における小脳顆粒細胞のエレクトロポレーション法による遺伝子導入の子宮内を使用して

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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 ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.