ラボでの作業は、NSF（MCB0919933）とNIH（GM088790）によってサポートされています。

1。トランクのフラグメントの再生アッセイ<ol><li>動物を確保するために再生アッセイ前に少なくとも5日間ワームのコホートを供給停止は、廃棄物や摂取した食べ物（〜30そのままワーム、それぞれのワーム〜8〜10ミリメートル長いポスト飢餓）は無料です。 </li><li>検定の日に、水で事前に冷凍水平に氷皿を洗い、ラップで平らなアイス表面を覆う。鉗子を使用して、ラップで1つろ紙を置きます。 </li><li>春の数滴の水でろ紙を湿ら。トランスファーピペットを使用して、フィルタ（フィルタあたり&lt;20ワーム）の上にプラナリアを転送します。ワームは、より多くの温泉水でrepipettingで、必要に応じて、フィルタを再配置することができます。余分な水分を取り除きます。 </li><li>メスを使用して、動物や咽頭の前端（ 図1A）の前方頂点間のほぼ中間に作られたシングルカットで頭部を切断する。 </li><li>メスを使用して、約半分尾の動物の終わりと咽頭（ 図1A）の後端との間に作られたシングルカットで尾部を切断する。カットした後70％エタノールで湿らせたペーパータオルを使用してメスで余分な粘液を取り除く。メスから余分なエタノールを拭きます。 </li><li>春の水を含むペトリ皿（100 × 25ミリメートルの深さ）にピンセットを介してフィルタを転送する。 （創縫合のための、傷のピンチと暗くによる観測）〜3分間待ちます。 </li><li>ろ紙からの回生アッセイと恒温インキュベーターへの転送（24℃）の所望の培地を含むペトリ皿にトランクの断片をすすぎます。 </li><li>再生の構造は（ 図1A）識別可能〜1週間後にスコアが再生表現型、。 </li></ol> 2。プラジカンテルにより誘導される二極化：再生の薬理学的操作<ol><li> DMSOに可溶化薬剤（200mmの株式に対して1mLので62.4mg PZQ）によってプラジカンテル（PZQ）株式を準備します。同じ日に使用してください。 </li><li>バッファMontjuch塩の薬物含有溶液（90μMPZQ）の50mLのを確認。分散性を確保するために〜1分間ボルテックス。 </li><li>最後のステップ[1.7]でPZQにトランクのフラグメントのコホートを公開する、[プロトコル＃1]上記の詳細として、トランクの断片回生アッセイを実行します。 </li><li> 24-48時間後に、交換PZQ含有、湧水のためのメディアをワームのいくつかの（&gt; 3）回洗浄。 </li><li>インキュベーターにワームを返します。スコアの二極化（二頭、 図1B）切断後一週間。 </li></ol> 3。再生の遺伝子操作：in vivoでのRNAi送りプロトコルで <ol><li>アッセイ前に、鶏の肝臓ホモジネートを準備。 foodstockから脂肪、黄色のセクション、ピューレ、残りの肝臓を捨てる。ストレージのためのメッシュのストレーナー、アリコートの懸濁液（1.5mlのチューブ、-20 ° C）を介してフィルタピューレ。 </li><li>アッセイ前に、-20℃で保存され、1mLの試料に供給を小分けでウシ赤血球（RBC）を準備する</li><li> RNAiのワームを選択します。 3つのコホートが使用されています：目的の遺伝子、ポジティブコントロール（既知のRNAi表現型を有する遺伝子; 図1C、D＆E）とネガティブコントロール（無RNAiの表現型を有する遺伝子、実験的なコホートと比較してノックダウンレベルの評価にも有用）。各コホートの場合は、〜250ワームを（少なくとも5日間飢え後に長い〜8〜10ミリメートル）を使用します。プラスチック製の浴槽で温泉水に保管してください。 </li><li>興味のdsRNA標的遺伝子を発現する大腸菌の各RNAiコホート、雪解けストックの[8]、鶏の肝臓ホモジネートのチューブと一緒に[3.1]と赤血球のチューブ[3.2]。 </li><li>ペレット細菌（13,000 G、1分）。 2xYTメディア（〜700μl）で上清とペレットを再懸濁しますを削除します。 Recentrifugeは、氷の上清、場所ペレットを捨てる。 </li><li>チップの先端をカットすることで大口径P200のピペットチップを作成します。徹底的にRNAiの供給ミックスを作成するために鶏肝臓ホモジネート（150μL）と赤血球（50μL）の混合物で、細菌ペレットを再懸濁します。遠心により気泡を取り除きます。 </li><li>給餌の場合は、ワームを（〜1インチの深さを残す）を含むプラスチックの浴槽からの水の大部分を取り除く。このコンテナの中心にワームを集中させるスワール浴槽、およびワームをcorralling円でピペットのRNAi給餌ミックス。慎重に他の食事を乱すことなく、RNAiの給餌ミックスに戻って脱出を同軸ケーブルに転送ピペットを使用してください！ </li><li>給餌（〜1時間）した後、よく飼育プラナリアを識別する。これらのワームは、摂取した赤血球から深い赤の着色を示す。他人を捨てる。 </li><li>慎重に、新鮮な湧き水を使用してソリューションを供給する食品の排せつ物を防ぐために、妨害を最小限に置き換えます。これを行うには、静かに濁った水を注ぐ、転送ピペットを用いてチューブの片側にワームをローカライズし、慎重に新鮮な水で置き換えるには、浴槽の反対側の壁を注いだ。再び潜水するのプラナリアは、すぐに空気に長時間さらされるとしても、食品の排せつ物をもたらす。 </li><li>疎SEらの容器のふたやインキュベーターに復帰（24 ° C） </li><li>再生サイクル[プロトコル＃1]のRNAi表現型をスクリーニングするためのを織り交ぜ、複数のサイクルにわたるRNAiの給餌プロトコル（離れて2〜3日間）、繰り返します。多くの遺伝子のための効果的な給餌と再生のための標準プロトコルは、継続時間の合計で1ヶ月〜かかる図2に示されています。 </li></ol>最適なプロトコルはこのようなmRNAの安定性、組織局在性、タンパク質のperdurance、または再生後に複数の給餌サイクルを排除する表現型の開発などの要因に左右されるように、異なる遺伝子のためにこのスキームを変更します。単にネガティブコントロールのコホートで標的mRNAレベルを比較する表現型の浸透率（見かけの場合）のため、または定量PCR手法でスクリーニングすることによって、ノックダウンのレベルを評価する。 4。代表的な結果： トランクのフラグメントの再生アッセイでは、すべてのワームは、通常の前後（&quot;尾に頭&#39;）極性で再生成する必要がありますように堅牢です。これは、前eyespotsの出現（asterixed、 図1A）により促進、カットした後五日するとすぐに得点することができます。しかし、失われた構造物の完全な形態学的な復元は一週間後に発生します。双頭のワームを得るためにPZQによるトランクの断片の再生の薬理学的操作（ 図1B）も堅牢である（EC 50 = 87（+ - ）11％バイポーラフラグメント、48時間2070μMPZQ）。二極化は、単一の再生サイクルを介して行われます。これらのアッセイの下の有効性は、おそらく薬の溶解性および/またはストレージに関する問題、またはPZQは効果がない別の扁形動物の種の使用にも関する。低浸透率を有する薬剤の場合は、anteriorizationのインデックスは、多くの場合、完全な二極22から脇の中間表現型を獲得するために使用されます。正の制御構文のRNAi実験に起因する表現型を図1に示されています： ニホンDugesiaのRNAi 六1（D J六- 1）目のない表現型23（ 図1C）、Dugesia ジャポニカPC2のRNAi（DJ - PC2）の結果光嫌悪反応24（ 図1D）とのRNAiの損失の結果Dugesiaスギβcatenin- 1（DJ -βcatenin- 1）トランクのフラグメント（ 図1E）から双頭の表現型16-19、21の結果。これらの表現型は、次のような単純なRNAiのプロトコルを使用して達成することができます：DJ -六1（FFxFx）、DJ - PC2（FFFx）、DJ -βcatenin- 1（FFxFx）、ここで、F =給餌サイクルとx =再生サイクルそれぞれ。 <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="図1"/> 図1：トランクのフラグメントの再生アッセイ左 、プラナリアの画像（上）と回路図（中央）切り出したトランクのフラグメントの位置を示すために右 、指示された時間（日数）で再生フラグメントの外観を示すトランクのフラグメントの再生のtimecourse B。。。 PZQの曝露によって生成される。双頭のプラナリアの画像DJ -六1のRNAi。モバイルコントロールのワームに比べて不動のDJ - PC2のRNAiワーム（ステンドグラス赤）の光嫌悪反応のDの損失、（ステンドによって生成されたC&#39;目のない&quot;ワーム緑）。Eバイポーラプラナリアは、DJ -βcatenin- 1 RNAiにより作り出した。 BEで、RNAiはワームのオリジナルの前端には、左に指向です。 <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="図2"/> 図2：D.における多くの遺伝子のノックダウンに有効である、複数の栄養（F）、及び再生サイクルを構成する典型的なRNAiのプロトコル（x）の供給と切断サイクルのシーケンスジャポニカ 。個々の遺伝子のためのこのプロトコルの変更が少ない（括弧）や給餌と再生のサイクルのより大きい数を使用する例については、最適な結果を得ることが必要になることがあります。

<ol>
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利害の衝突は宣言されません。

プロトコルは、 ジャポニカプラナリアDugesiaの再生を研究し、操作するためここに詳細のアッセイを説明した。彼らは単純なものであり、それらは容易に実験室や教室で行うことができるというような特殊な機器を必要としません。アッセイは個別に実行、または組み合わせて（in vivoでの薬物の効能の化学遺伝学的スクリーニングのための）と候補遺伝子レベルで行うことができる、または公正な、より高いスループットスクリーニング8に適応可能であることができる。自分の右側にプラナリアの興味深い生物学を研究するためかどうか、または組織再生を研究するための代替モデルの哺乳類ホモログの in vivo機能で評価するために、これらのアプローチは、研究者の多様な範囲からの関心を触媒してください。

<table border="1"><tbody><tr><th>試薬</th><th>ベンダ</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>春の水</td><td> Kandiyohi。プレミアムウォーターズ株式会社ミネアポリス、ミネソタ州</td><td> N / A </td><td>温泉水の他の形態も良好に動作します。生存率アッセイで初の試み。 </td></tr><tr><td>塩化ナトリウム（1.6mm厚）、塩化カルシウム（1mMの）、硫酸マグネシウム（1mMの）、MgCl2の（0.1）、塩化カリウム（0.1）、NaHCO3水溶液（さ1.2mm）、HEPES（1.5mmの）：1 xは、モンジュック塩をバッファ。 24でpH7.4の℃の</td><td>複数のベンダー</td><td> N / A </td><td> pH緩衝性を確保するために再生アッセイ中の薬物治療のための人工的な水。 5月8日Holtfreterのソリューションは、代替手段です。 </td></tr><tr><td> 2xYT培地</td><td>フィッシャーサイエンティフィック</td><td> BP2467 - 500 </td><td>メディア= 31グラム/ L。オートクレーブ。 </td></tr><tr><td>ペトリディッシュ（100x25mm） </td><td>フィッシャーサイエンティフィック</td><td> 08-757-11 </td><td>再生サイクルの間に住宅のワーム</td></tr><tr><td>スクエアディッシュ（100x100x15mm） </td><td>フィッシャーサイエンティフィック</td><td> 08から757 - 11A </td><td>ワーム切断面として使用される製氷皿に水、凍結で埋める</td></tr><tr><td>プラスチック製の浴槽：ジップロックツイスト&#39;N LOC（16オンス）。 </td><td>様々な小売業者</td><td> N / A </td><td> RNAiのコホートに便利な防水容器</td></tr><tr><td>鶏のレバー</td><td>商業用食料品</td><td> N / A </td><td>抗生物質の回避により、有機供給に向けてバイアス、。 </td></tr><tr><td>ハンドブレンダー</td><td>あらゆる台所のサプライヤー</td><td> N / A </td><td>鶏レバーのピューレを作るために使用</td></tr><tr><td>ワイヤー1ミリメートルメッシュストレーナー</td><td>あらゆる台所のサプライヤー</td><td> N / A </td><td>鶏レバーのピューレに負担をかけるために使用</td></tr><tr><td>ウシ赤血球</td><td> Lampire生物研究所</td><td> 7240807 </td><td> 100パーセントRBC懸濁液を洗​​浄し、プール</td></tr><tr><td>円形のろ紙</td><td>ワットマン＃3 </td><td> 1003 055 </td><td></td></tr><tr><td>注入器</td><td>フィッシャーサイエンティフィック</td><td> 13-711-41 </td><td></td></tr><tr><td>滅菌、外科ブレード</td><td>複数のベンダー</td><td> N / A </td><td></td></tr><tr><td> ±プラジカンテル</td><td>シグマアルドリッチ</td><td> P4668 </td><td> 4℃で暗所に保管して粉体のアリコート℃の生気を奪う。 </td></tr><tr><td> DJ六- 1 </td><td></td><td> GenBankのAJ557022.1 </td><td> &quot;目のない&quot;表現型は23 RNAi実験のポジティブコントロール</td></tr><tr><td> DJ -βcatenin- 1 </td><td></td><td> GenBankのAB181913.1 </td><td>双頭の表現型17のRNAi実験のポジティブコントロール</td></tr><tr><td> DJ - PC2 </td><td></td><td></td><td>羞明の表現型24の損失のためのRNAiのポジティブコントロール</td></tr></tbody></table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

自由生活プラナリアの扁形動物は彼らの驚くべき再生能力を1から実験的な利用等により、長い歴史を持っている。これらの動物から摘出された小さな断片が欠落しているボディ構造の再生に続くオリジナルのボディプランを改革。 &quot;トランク&quot;の断片がそのままワームから切断されている場合、例えば、新たな&quot;頭は&quot;前方に生成されると&quot;尾&quot;は（ 図切断する前の元のボディ構造の&quot;頭部と尾部&quot;極性を復元後方に再生成されます1A）。 再生は通常の身体の恒常性と強制組織の再生中に〜30の異なる細胞型に分化&quot;neoblasts&quot;として知られるプラナリア幹細胞、によって駆動されます。この再生過程では、実証するため、強力かつ簡単です。いくつかの先駆的なラボの献身のおかげでは、多くのツールと​​機能的遺伝学的手法は、現在このモデルシステムに最適化されています。その結果、かなりの最近の進展は、プラナリアの発達可塑性2-9を支える分子事象を理解し、操作で行われている。 プラナリアのモデルシステムは、科学の広い範囲に興味を持ってもらえるでしょう。神経科学者のために、モデルは単純に全体の神経系の再生ではなく、通常は多くの確立されたモデルでの研究の焦点となっている単一の神経細胞のプロセスの再生/修復を勉強する機会を与えてくれます。プラナリアは、神経伝達物質10の茄多を表現する中枢神経系11、12の進化を研究するための重要なシステムを表しており、行動のスクリーニングの可能性13、14を持っている。 再生結果は、薬理学的および遺伝的apparoachesによる操作に適している。例えば、薬は単に切断後の異なる時点で薬剤を含む溶液中で身体の断片を配置することによって、再生への影響についてスクリーニングすることができる。個々の遺伝子の役割は、マイクロインジェクションのサイクルを介して、または細菌で発現された二本鎖RNAの構造8、9、15を供給することにより、どちらかを達成することができるノックダウン方式（ 生体 RNAi の ）、使って研究することができます。どちらのアプローチは、バイポーラの動物16から21までの高い浸透率、たとえば、再生時に視覚的に印象的な表現型を生成することができます。このようなメソッドのこのモデルと実装の ​​採用を促進するために、我々はプラナリアDugesiaはジャポニカ使用して薬理学的および遺伝学的ア ​​ッセイ（in vivoでのRNAi の摂食による）のためのこのビデオの記事プロトコルで披露。

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

生きた動物の中で幹細胞の分化を研究するための魅力的なモデルは、プラナリア扁形動物です。再生は簡単に基本的な実験室で行われる単純な切断実験により研究し、薬理学的および遺伝的（に適しているさ<em&gt; in vivoで</em&gt; RNAi）は、この記事ではプロトコルで詳述するように操作。

プラナリアの再生を操作するために薬理学的および機能的遺伝学的アッセイ<emジャポニカ> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

pharmacological-functional-genetic-assays-to-manipulate-regeneration

Research

JoVE Journal

Biology

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

36.7K ビュー.  University of Minnesota Medical School. 生きた動物の中で幹細胞の分化を研究するための魅力的なモデルは、プラナリア扁形動物です。再生は簡単に基本的な実験室で行われる単純な切断実験により研究し、薬理学的および遺伝的（に適しているさ in vivoで RNAi）は、この記事ではプロトコルで詳述するように操作。

ビデオ: Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

マウス用ダーク/ライト遷移テスト

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

生物学

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

皮質の神経形成：研究室の進歩からセルベースの​​治療への移行

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

鶏胚における神経誘導のためのアッセイ

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

マウス用高架式十字迷路

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

成体マウスから海馬歯状回の解剖

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

胚形成の間に機能的な冗長性をテストするための逆遺伝学的アプローチ

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

遺伝的関連の機能的基準をテストするための対立遺伝子特異的遺伝子発現アッセイ

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

プラナリアの固定化、部分照射し、組織移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

細菌機能ネットワークとPathwaysでのマッピング<em&gt;大腸菌</em&gt;合成遺伝子配列を用いて

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

遺伝子発現用機械の機能的アッセイのための小規模の核抽出物

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

プラナリアの再生を操作するために薬理学的および機能的遺伝学的アッセイ Dugesia