CBMに：この作品は、&quot;逆遺伝学的アプローチゼブラフィッシュゲノムを耕し、&quot;NIH R01 HG002995によってサポートされていました。

パート：精子凍結ソリューション 1。ギンズバーグフィッシュリンガー（3日ごとに新鮮な確認、ギンズバーグ、1963） 注：添加の順序は、沈殿を防止することが重要です 450にmlの滅菌ddH2Oに溶解する： <ul><li>塩化ナトリウム3.25グラム</li><li>塩化カリウム0.125グラム</li><li>塩化カルシウム•2 H2O 0.175グラム その後、追加します。 </li><li> NaHCO3水溶液0.10グラム</li></ul> 4℃で滅菌ddH2Oと店舗℃で500 mlに最終容量をもたらす 2。凍結培地（新鮮な毎日を作る） <ol style="list-style-type:lower-roman"><li>メタノールなし<ul><li>ギンズバーグの魚は10mlリンゲル（室温を。） </li><li>脱脂粉乳1.5 GM </li></ul></li><li>メタノールで 注：添加の順序は、沈殿を防止することが重要です <ul><li>ギンズバーグの魚は9ミリリットル（室温を。）リンガーズ</li><li>メタノール1ミリリットル</li><li>脱脂粉乳1.5グラム</li></ul> 凍結媒体を組み立てた後、20分間よく混ぜる。オービタルシェーカーまたはロッカーで。ご使用になる前に、1mlのエッペンドルフチュー​​ブに分注し、表面の気泡を回避。 </li></ol>パートB：精子の凍結に必要な材料精子の凍結に必要な材料<ol><li> 10μlの使い捨てピペット（Fisherbrandの猫＃22から358697） </li><li> MESAB / Tricaineを含む魚の水の250ミリリットルのビーカー</li><li>時計メガネ（パイレックスの猫＃9985〜75） </li><li> P20 pipetman（または同等）とヒント</li><li>スポンジの魚ホルダー（スクイーズしながら男性を保持する） </li><li>上記の舞台照明付き実体顕微鏡</li><li>フラットピンセット（例えばミリポアタイプ） </li><li> 2.0ミリリットルクライオバイアル（コーニング猫＃430488） </li><li> 10 × 10 cryoboxes（Nalgene猫＃03から337 - 7AA） </li><li>発泡スチロール容器は、細かく破砕乾燥ice1の〜15 Cmを埋め</li><li> 15 mlコニカルチューブ（ファルコン＃352099） </li><li>液体窒素を含有するデュワーフラスコ</li><li>長いトング（例：CMSフィッシャーヘルスケア猫＃10 - 316B） </li><li> Cryogloves </li><li>男性のための回収タンク</li><li>メタノールなく凍結媒体を充填した1エッペンドルフチュー​​ブ</li><li>メタノールによる凍結培地を充填した1エッペンドルフチュー​​ブ</li><li> Cryofreezer（例えばテイラー - ワルトンK -シリーズ） </li></ol> 1細かく砕いたドライアイスを作るための簡単な方法は、タオルでそれをラップし、ハンマーで粉砕することです。より効率的な方法は、ドライアイスのグラインダー、たとえばクローソンモデルRE - 2を使用することです。 パートC：精子凍結法<ol><li> キャピラリーチューブをマーク：前初めに、（底部からすなわち16.7ミリメートルを、図を参照してください1.1。）3.33μlのレベルで研究室のペンで10μlのキャピラリーチューブをマーク。 </li><li> 男性の麻酔：MESAB / tricaineを入れたビーカーに配置男性（s）は魚の水（&quot;ゼブラブック&quot;のレシピ）で希釈し</li><li> DRY FISH：一度麻酔は、プラスチックのスプーンを使用して麻酔薬の魚アウトを持ち上げ、ゆっくりと腹側を乾燥するために特別な注意を払って、ペーパータオル上で乾燥した魚を汚点。それは徹底的に排出腔の周囲に乾燥することが重要ですので、水は精子を活性化する。スポンジホルダーの位置の魚、腹側を上（図1.2）。しりびれ周辺地域は、依然としてキムワイプで軽く乾燥し、湿ったである場合。途中で精子を押さないように注意してください！ </li><li>場所は、キャピラリーチューブに含まれているゴム製の口のピペットアダプタにキャピラリチューブをマーク。アダプタは一端のマウスピースや他のオンキャピラリーホルダーを持っているゴム製のホースです。また、キャピラリーチューブは、適切な直径のタイゴンチューブの短い断片を経由して50μlのハミルトンシリンジに接続することができます。 </li><li>範囲で男性を置き、慎重に離れて毛細管の端を使用して骨盤のフィンを分散することで、泌尿生殖器の開口部を公開します。 </li><li> 精子を収集します。穏やかに滑らかな鉗子（例えば、ミリポア社）で魚の側面をなでることによって精子を排出するか、そっと人差し指と親指の間に魚の側面を絞って。それは穏やかな吸引（図1.2）を使用して追放されているキャピラリーチューブで精子を回収します。精子で追放されることがあります糞を避けてください。 </li><li> μlを3.3〜精子の音量を標準化：精子の下流のボリューム、または、キャピラリチューブ（すなわち16.7ミリメートル以上）のペンのマークを超えている場合は、手順8に進みます。精液の容積は、ターゲットボリュームに到達しない場合には、メタノール（図1.3）することなく凍結培地を使用してペンのマークに音量を正規化する。許容される精子の最小量は、精子の質によって異なります。良い精子は白と不透明です。悪い精子は水っぽく見えます。 1μlの良い精子（または1 / 3目標）と2μlの悪い精子（または2 / 3ターゲット）：一般的に、我々は最小値として受け入れます。ボリュームは現在3.3μlに正規化されます。 </li><li> 精子を凍結保護剤を追加：Mで凍結培地を吸い取るオレンジ色のマーク（。。図1.4約総量は現在20μlの場合）にエタノール。気泡を導入しないように特別な注意を払って、時計のガラスのきれいな部分に精子と凍結保護剤混合物​​を排出する。穏やかにピペッティングして混合し、ダウン〜4回（気泡を導入しないように）。 </li><li> 2 CRYOVIALSのそれぞれに10μlの精子を置きます：精子のピペットで10μlの：関連情報（図1.5）で標識された2つの別個のcryovialsの底部に凍結保護剤のソリューション。 、すぐにキャップバイアルを移動し、室内温度15 mlのFalconチューブ一クライオバイアル/チューブの底にそれらをドロップします。キャップファルコンチューブと砕いたドライアイスにクライオバイアル含有ファルコンチューブのペアを挿入します。 </li><li> 20分間の精子を凍結。ドライアイス上で：ドライアイスは、微粉末状ではなく、ペレットにする必要があります。チューブは、十分に深くだけ彼らのキャップが（図1.6）を示すことがドライアイスに挿入する必要があります。ドライアイスでチューブを追跡するためには、1-20（キャップ​​に書かれた）から、ファルコンチューブの各ペアの数を与えると彼らはドライアイスに入る時間を記録する。 </li><li> 液体窒素に会場のCRYOVIALS：。精子は20分間ドライアイスで凍結された後は、液体窒素含有デュワーフラスコに移す。精子は、液体窒素の冷凍庫に配置する時までここに保存されます。試料の温度が上昇しないように、液体窒素の風呂で冷凍ボックスに配置すること、場所のフリーザーボックス。デュワーフラスコからバイアルを回収し、cryoglovesで処理するための長い金属製のはさみを使用してください。冷凍庫ボックスは発泡スチロールの容器に液体窒素に浸漬保たれていれば別の方法として、cryovialsは冷凍庫のボックスにドライアイスから直接転送することができます。 </li><li> 極低温の液体窒素の冷凍庫に保管してcryovialsは長期 。精子の生存能力を維持するために、それは、バイアルを液体窒素に浸漬保存されていることが重要です。我々の経験では、気相で保存されているバイアルは、時間の経過とともに生存能力を失う。 速度が重要です！凍結保護剤を追加し、ドライアイス（すなわちステップ6-10）で、精子のバイアルを配置するまでの時間は最大30秒にする必要があります。 </li></ol>パートD：凍結保存精子を用いた体外受精の方法我々は、これが二人で最良に動作することを見つける。他の人が精子を解凍しながら一人は女性から卵子を圧縮します。 <ol><li> 33℃に水浴を設定する</li><li>雪解けへの準備ができるまで、液体窒素の冷凍庫と転送から液体窒素含有デュワーフラスコにクライオバイアル取り外します。 </li><li> 35ミリメートルのプラスチックシャーレに女性から卵を絞り出します。可能であれば、卵の3クラッチを取得し、一皿に結合してみてください。あなたの最初のクラッチの1分以内に3つの良いクラッチを得ることができない場合は、（一つの良いクラッチが十分である）ステップ4に進みます。良いクラッチの定義：&gt; 150均一に見栄えの良い卵（すなわち黄色、劣化の指標のない白い破片を持つ）。精子を融解している間覆われて皿にしてください。 </li><li>液体窒素からバイアルを取り外し、キャップを取り外します。 33 80から10秒° Cのウォーターバスにすぐに浸す。バイアル〜1 / 2の方法。 </li><li>素早く上下にピペッティングによりバイアルとのミックスを70μlを室温ハンクスを追加。すぐに卵に追加して、ピペットの先端で軽く混ぜる。 </li><li>遅滞なく、750μlの魚の水を加えて精子と卵子を活性化する。ミックスに渦巻く。室温で5分間インキュベートする。 </li><li> 5分後、28で魚の水とインキュベート皿に料理を埋める℃に2-3時間後、数えると100ミリメートル料理（50/dish）に肥沃な胚を移す。受精の頻度を決定することができるように無精卵を数える。 </li><li>幼虫の世話をする！最初の5日間は、毎日水を変えると死亡胚を削除します。 5日目の保育園に膀胱を泳ぐいるだけ幼虫を置く。 </li></ol>パートE：代表結果我々は2001年と2003年の間にティリングのために8600凍結精子サンプルのライブラリを作成するこのcrypreservationの方法論を使用。我々は、これらのサンプルの106を解凍し、その稔性を決定した。出生率は凍結精子で受精した卵の総数で割った値で体外受精で生産可能な胚の比率です。新鮮な（cryporeserved以外）精子一般的に&gt; 95％の稔性がある、凍結保存した精子は、非常に低い出生率を持っている：29％（N = 106バイアル）の平均を。出生率のバイアルからバイアルの変動は1％未満から62％と高いに至るまで、大きいです。このばらつきは、図の標準偏差を表す大きなエラーバーで表されます。 2。しかし、これまでに融解106精子のサンプルで我々は一度だけ、両方の精子サンプルで0％の肥沃度を経験し、対応するティリングの突然変異の回復に失敗しました。 数年間のコース（2001-2008）を介して我々のライブラリから精子バイアルを融解することによって我々はこの方法で凍結精子のサンプルの平均出生率が残っていることを発見した（図2）時間の経過とともに安定した。 2001年に融解した凍結精子のサンプルの平均稔性は23.5％（11精子のサンプルに基づく）であり、2008年には25％（21精子のサンプルに基づく）であった。 他の研究者の数は今や彼らのラボでこの精子凍結プロトコルを使用しています。いくつかは我々が観察したものと同様の出生率とサンプル間の変動との良好な成功を報告している。その他の報告によれば、このプロトコルは、彼らの手で動作させるために失敗している。私たちは、問題のありそうな情報源があることを信じて： <ol><li> ;時にドライアイスで冷却の異なる速度につながる、我々はプロトコルで推奨されている正確なcryovialsまたはコニカルチューブを使用するために失敗</li><li> 、次善の冷却速度につながる、粉末ドライアイスを使用して失敗</li><li>貧しい肥沃でオスの魚を使用：単男性から精子の少なくとも1μlを取得する必要があります。それは100％の肥沃度近く得ることは非常に少ない新鮮な精子を取るために、また新鮮な精子で体外受精を行うことによって男性の生殖能力をテストすることができます、しかしこれは誤解を招く可能性があります。 </li><li>体外受精でのサブ標準的な品質の卵を使用した。決して完璧なクラッチを使用すると常に0％の肥沃度近くにも、実際に良い肥沃度を（重複バイアルの稔性に基づいて）持っている精子のサンプルからにつながります。 </li></ol><img src="/files/ftp_upload/1395/1395fig1.jpg" alt="図1" /> 図1。精子凍結方法論の概略図。 <img src="/files/ftp_upload/1395/1395fig2.jpg" alt="図2" /> 図2凍結保存精子の受精能は、時間をかけて安定している。パーセントの肥沃度は凍結精子で受精した卵の総数で割った値で体外受精で生産可能な胚の数です。 &quot;n&quot;は、生殖能力に対応する年間でテストされて凍結保存した精子のバイアルの数を指します。エラーバーは標準偏差を表しています。各精子のバイアルは、別の男性からなので、バイアルからバイアルの変動が観察される。

<ol>
	<li>Ginsburg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm-egg+association+and+its+relationship+to+activation+of+the+egg+in+salmonid+fishes.">Sperm-egg association and its relationship to activation of the egg in salmonid fishes.</a> J. Embryol. Exp. Morphol. 11, 13-33 (1963).</li><li>Harvey, B., Kelley, R. N., Ashwood-Smith, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+zebra+fish+spermatozoa+using+methanol.">Cryopreservation of zebra fish spermatozoa using methanol.</a> Can. J. Zoology. 60, 1867-1870 (1982).</li></ol>

他の研究者の数は今や彼らのラボでこの精子凍結プロトコルを使用しています。いくつかは同じような肥沃度と我々が観察した同じサンプル間の変動を報告している。その他の報告によれば、このプロトコルは、彼らの手で動作させるために失敗している。私たちは、問題のありそうな情報源があることを信じて： <ol><li> ;時にドライアイスで冷却の異なる速度につながる、我々はプロトコルで推奨されている正確なcryovialsまたはコニカルチューブを使用するために失敗</li><li> 、次善の冷却速度につながる、粉末ドライアイスを使用して失敗</li><li>貧しい肥沃でオスの魚を使用：単男性から精子の少なくとも1μlを取得する必要があります。それは100％の肥沃度近く得ることは非常に少ない新鮮な精子を取るために、また新鮮な精子で体外受精を行うことによって男性の生殖能力をテストすることができます、しかしこれは誤解を招く可能性があります。大きく、より確実に色付きの男性は一般的に、より高品質の精子を与える。 </li><li>体外受精でのサブ標準的な品質の卵を使用した。決して完璧なクラッチを使用すると常に0％の肥沃度近くにも、実際に良い肥沃度を（重複バイアルの稔性に基づいて）持っている精子のサンプルからにつながります。 </li></ol>

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>10 &mu;l disposable pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-358697</td>
<td>&nbsp;</td>
<tr>
<td>Watch glasses</td>
<td>&nbsp;</td>
<td>Pyrex</td>
<td>9985-75</td>
<td>&nbsp;</td>
<tr>
<td>2 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430488</td>
<td>&nbsp;</td>
<tr>
<td>10 x 10 cryoboxes</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>03-337-7AA</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>Tongs</td>
<td>&nbsp;</td>
<td>Fisher Health Care</td>
<td>10-316B</td>
<td>&nbsp;</td>
<tr>
<td>Cryofreezer</td>
<td>&nbsp;</td>
<td>Taylor-Warton</td>
<td>K-series</td>
<td>For example</td>
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a high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization

これはハーヴェイの方法（ハーヴェイら、1982）の適応であるゼブラフィッシュの精子の凍結保存のための方法です。我々は両方の手順を合理化し、サンプルの均一性を高めること、元のプロトコルには2つの変更を導入している。まず、凍結保護剤を含まない凍結​​メディアを使用して、すべての精子の量を正規化する。第二に、凍結保存精子の代わりに毛細管のcryovialsに格納されています。 （δ° C /時間）凍結融解精子の率は、おそらくこの手順で制御するには二つの最も重要な変数です。このような理由から、指定されたもののために別のチューブを代用しないでください。 2のチームで作業すること約2時間でチームあたり100人の男性の精子を凍結することが可能です。精子は、このプロトコルを使用すると25％の肥沃度の平均を持っている（受精卵の総数で割った値で体外受精で生成された実行可能な胚の数として測定される）、このパーセントの出生率が長年にわたって安定な凍結保存。

Watch this Scientific Journal Video about A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization at JoVE.com

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

これは、ゼブラフィッシュのためのハイスループット精子の凍結保存プロトコルです。精子は、このプロトコルを使用すると、その後の体外受精で25％の肥沃度の平均値を持ち、長年にわたって安定な凍結保存。

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

a-high-throughput-method-for-zebrafish-sperm-cryopreservation-vitro

Research

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Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

30.8K ビュー.  University of California, Davis. これは、ゼブラフィッシュのためのハイスループット精子の凍結保存プロトコルです。精子は、このプロトコルを使用すると、その後の体外受精で25％の肥沃度の平均値を持ち、長年にわたって安定な凍結保存。

ビデオ: A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

生物学

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

ヒトmRNA前駆体上にマッピングリボ核の迅速なハイスループット法（RNPS）

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

アイスキャップ：成長させる方法シロイヌナズナ</emハイスループットジェノタイピング用96ウェルプレートで>とトマト

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I ·クライオキットを使用してマウスの精子の凍結保存と回復

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

半数ゼブラフィッシュ胚の生成により<em&gt;体外</em&gt;受精

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

ゼブラフィッシュ胚を用いた高スループットへのアプローチマニュアル小分子スクリーン

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...