、スラッシャーの研究基金からの新しい研究賞、ボストン小児病院で心臓伝導基金への拠出、この作品は、国立衛生研究所から研究助成金（HL088206 HL068915）によってサポートされています。

パート1：鋳型を構築アセンブル<ol><li>シリコンチューブ（VWR）を半減、3 cmの長さの部分にカットするためにカミソリの刃を使用してください。 </li><li>チューブの各端の内側にインプラントグレードのRTVシリコーン接着剤（ローディア社）のドロップを置きます。 </li><li>すぐにシリコーン接着剤のドロップにポリエステルメッシュ（マクマスター - カー）の小片（1センチメートル× 1cmを）置き、チューブの端に合わせて取り付けます。これは、構造、添付ファイルのためのわずかに隆起し、平坦な表面を提供します。もう一方の端について、この手順を繰り返します。 </li><li>金型は3日間室温で乾燥することができます。これらは基本的に使い捨てなので、時間20〜30金型を作っておくと便利です。 </li><li>接着剤が完全に乾燥したら、必要になるまで、70％エタノールを入れたビーカー内でこれらの型を格納します。このステップでは、カビを殺菌するのに役立ちますし、完成品を図1に示されています。 </li></ol>パート2：筋芽細胞を分離するための準備<ol><li> 250に1 mgのラミニン（シグマ）を希釈液0.22μmのはフィルター滅菌リン酸緩衝生理食塩水（PBS）は、1％ペニシリン/ストレプトマイシン（Invitrogen社）と1％のFungizone（Invitrogen）を含む。 </li><li> 4手順1.1から希釈ラミニン溶液1mlと主要骨格筋芽細胞を播種する前に少なくとも4時間、37℃でインキュベートするとコート150mmの組織培養プレート（BDファルコン）。 </li><li> （20％FBS（アトランタバイオロジカル）、5 ng / Lの塩基性線維芽細胞増殖因子（プロメガ社製）、1％ペニシリン/ストレプトマイシン（Invitrogen社）、及び1％FungizoneとハムのF - 10栄養混合物（Sigma）を混合することにより、筋芽細胞の培地を作るインビトロジェン）。 </li><li> 100mLの2.4 U / mLのディスパーゼ- 2（Roche社）で〜295 U / mgのコラゲナーゼ2（ワーシントン）1gを溶解することにより骨格筋の消化溶液を調製。 0.037グラムのCaCl 2を加え、よく混ぜる。 -20℃で10mLのシリンジとストアのアリコートに装着0.2μmのフィルターディスクを使用して消化バッファーを滅菌フィルタリング° C筋芽細胞のメディアと一緒に37組織の消化に必要な量を置きます。 </li></ol>パート3：骨格筋からの筋芽細胞の分離<ol><li>鉗子と小さなハサミを用いて、脊髄に沿って背中を下にスライス皮膚と筋膜層に戻って剥離し、筋肉を切り出すことによりデッド新生児Lewisラットから傍脊柱筋を取り除く。 </li><li>組織培養フードの中で、氷上で1 ×ハンクス平衡塩溶液の40 mLの（HBSS）（Invitrogen社）を充填した50mLのコニカルチューブ（BDファルコン）で摘出した筋肉を置きます。筋肉片がチューブの底に沈降し、慎重に文化のフード（ベイカー）で滅菌パスツールピペットを用いて真空吸引によって液と血液の残りの大部分を除去することができます。 </li><li> 150 mmの培養皿上に回収筋肉片を注ぎ、吸引で可能な限り残留液をできるだけ多く削除します。 2個のシングル両刃かみそりの刃を使用すると、厚いペースト状に組織をミンチ。ペーストを予め温め消化溶液を注ぐと10 mLのピペットを使用して新鮮な50 mlチューブに混合物を移す。酵素消化中に、組織は時折コードレスピペットエイド（ドラモンド）の上に取り付けた10 mLのピペットを用いて（バブリングなし）摩砕しています。再び、組織が余分な液体を解決し、削除することができます。 </li><li>ロッキングプラットフォーム上にチューブを置き、約30分間37℃で消化。 </li><li>筋肉が液化されると、600 XG（ベックマンGP）で10分間室温で70μmのセルストレーナー（BDファルコン）と遠心分離によるソリューションを渡します。パスツールピペットや吸引で、できるだけ液体の限りを削除し、暖かい筋芽細胞の培地の10mLでペレットを再懸濁します。 </li><li>プレ板15分間コーティングされていない150mmの組織培養プレート上で細胞が線維芽細胞を取り除く手助けをする。 </li><li>まだラミニンコートculturewareに接続されていると、1〜2仔ラット用のプレートがあるように希釈していないセルを転送する。 37 ° Cで5％CO 2の加湿インキュベーターセットにプレートを置きます。 </li></ol>第4部：鋳造工学、組織構造（5mL）を<ol><li> 1〜2日後、、インキュベーターから150 mmの筋芽細胞の組織培養プレートの10を削除して37℃の1 × PBSで2回それらを洗い流して、各プレートに温かい0.05％トリプシン- EDTAを4mLを追加します。 5分間インキュベーターにプレートを返します。 </li><li>文化のフードにプレートから分離された筋芽細胞を収穫し、50 mlチューブ（BDファルコン）で液体を集める。プレートのすべてをリンスし、遠沈管内の細胞とこれを組み合わせることが筋芽細胞の培地10mLを使用してください。ペレットは600 xgで10分間室温で細胞（ベックマンGP）と、ステップ3.5で説明したように余分な液体を取り除く。 </li><li>氷の上に筋芽細胞の培地と場所溶液1.6mLに筋芽細胞を再懸濁します。 </li><li>鉗子を使用して、慎重にステップ1.5から4構造の金型を削除し、1X PBSで2回、それらを洗い流してください。 6ウェル組織のCuの各金型を配置する前にパスツールピペットでPBSのすべての痕跡から真空吸引ltureプレート（BDファルコン）。 </li><li>氷の上に構造の準備に必要な他の試薬 ​​を（10XハムのF10、抗生物質、1型コラーゲン[3.9 mg / mLの] [6]およびマトリゲル™）を収集し、37℃での筋芽細胞のメディアを保持する100μLペニシリン/ストレプトマイシン、100μLFungizone、1.6 mLのコラーゲン、15 mLコニカルチューブに750μLのマトリゲル™、500μL10XハムのF10を組み合わせる。 10秒、氷の上を10に設定ミニボルテックス（VWR）と場所を使用するためのソリューションを混ぜる。 </li><li>次に、ステップ3.5からステップ3.3から混合物を80のNaHCO 3のμLと筋芽細胞懸濁液を加える。 10秒間ボルテックスする全体の混合物を、気泡のソリューションをクリアするために数分間、氷のバケツにチューブを返します。 </li><li>それは10 mLのピペットを用いて固化し始める前に、金型に混合物を慎重にキャスト。各金型は、粘性液体の約1 mLを開催します。この手順の実行中に気泡を導入しないようにしてみてください。 </li><li>インキュベーターにキャストの構文を含むプレートを慎重に移す。 〜30分後、インキュベーターから固化したコンストラクトを削除し、慎重に組織構築をカバーするために各ウェルに筋芽細胞の培地を加える。インキュベーターにプレートを返す（図2）。 </li></ol>パート5：代表的な結果このプロトコルが正しく実行されると、筋芽細胞を含む組織の構造は、 生体移植のために（すなわち、金型から除去するとき）または2日後にin vitroでの解析でさらにできるようになります。 <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> 図1完成した構造型の例（ステップ1.5参照）。 <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> 図2。固化した筋芽細胞を含む組織には、[1]を構築する。 <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> 図3筋芽細胞を含む組織のH＆E染色切片の構築。 &quot;&quot;縦断面を表し、&quot;B&quot;は断面を示しています。

<ol>
	<li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169 (1), 72-85 (2006).</li><li>Rando, T. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+myoblast+purification,+characterization,+and+transplantation+for+cell-mediated+gene+therapy.">Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.</a> J Cell Biol. 125 (6), 1275-1287 (1994).</li><li>Blau, H. M., Webster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+muscle+cells.">Isolation and characterization of human muscle cells.</a> Proc Natl Acad Sci U S A. 78 (9), 5623-5627 (1981).</li><li>Powell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+human+bioartificial+muscles+expressing+a+foreign+recombinant+protein+for+gene+therapy.">Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy.</a> Hum Gene Ther. 10 (4), 565-577 (1999).</li><li>Vandenburgh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+skeletal+muscle+organoids+for+reversible+gene+therapy.">Tissue-engineered skeletal muscle organoids for reversible gene therapy.</a> Hum Gene Ther. 7 (17), 2195-2200 (1996).</li><li>Gallop, P. M., Seifter, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Properties+of+Soluble+Collagens.">Preparation and Properties of Soluble Collagens.</a> Methods in Enzymology. 6, 635-641 (1963).</li></ol>

動物実験は、ボストン小児病院での動物実験使用の委員会が定めるガイドラインおよび規則に従って行った。

組織の構造がキャストされるの金型は、任意の形状と大きさで行うことができますが、添付ファイルの少なくとも2点がなければなりません。そうでなければ、マトリックスと細胞は球状構造を形成し、細胞が死ぬ。現在のプロトコルでは、我々はこの目的のためにポリエステルメッシュを使用することを記載し、まだ我々はまた、正常にステンレスメッシュを使用している。明らかに、より...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Silicone tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60985-724</td>
<td>&nbsp;</td>
<tr>
<td>Silicone adhesive</td>
<td>&nbsp;</td>
<td>Rhodia Silicones</td>
<td>MED ADH 4300 RTV</td>
<td>&nbsp;</td>
<tr>
<td>Polyester Mesh</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>93185T17</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Nutrient Mixture F-10 HAM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N6908</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Fungizone</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15290-018</td>
<td>&nbsp;</td>
<tr>
<td>Dispase-2</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10295825001</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase 2</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>46H8863</td>
<td>&nbsp;</td>
<tr>
<td>Basic Fibroblast Growth Factor</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>G5071</td>
<td>&nbsp;</td>
<tr>
<td>150 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353025</td>
<td>&nbsp;</td>
<tr>
<td>0.05% (1X) Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>1X Hanks Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-112</td>
<td>&nbsp;</td>
<tr>
<td>7.5% NaHCO3</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25080-094</td>
<td>&nbsp;</td>
<tr>
<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>50 mL Conical Vial</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352098</td>
<td>&nbsp;</td>
<tr>
<td>15 mL Conical Vial</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>0.2 &mu;m filter</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>194-2520</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of myogenic engineered tissue constructs

電子ペースメーカーは、医療機器を救命しているという事実にもかかわらず、小児患者における長期的なパフォーマンスは、子供の小さいサイズと彼らの必然的な成長により課される制限には問題によりすることができます。その結果、心臓のリズム障害を有する小児患者のために特別に設計された革新的な治療のための真の必要性がある。我々は提案することを含むコラーゲンベースのマトリックスで構成される導電性の生物学的代替細胞が良く、成長に適応再発手術の必要性を削減し、大幅にこれらの患者の生活の質を向上させることができるautologously由来。本研究では、我々は、ファッションにハイドロゲルマトリックス内に心臓の上部および下部チャンバーの間の電気コンジットとして動作する外科的に移植可能な組織構造を主骨格筋芽細胞の細胞培養を組み込むための手順を説明します。最終的に、我々は、完全心ブロック児の房室の電気伝導を復元するために設計された組織のこのタイプを使用して期待しています。そのビューでは、我々は、確立されたプロトコルの修正バージョンを使用して、ラミニンコートした組織培養皿の上に新生児のLewisラットやプレート、それらの骨格筋から筋芽細胞を分離<sup&gt; [2、3]</sup&gt;。 1〜2日後、培養細胞は、抗生物質で1型コラーゲン、マトリゲル™、およびNaHCOを収集し、混合され<sub&gt; 3</sub&gt;。その結果、ほぼ任意の形状と大きさの金型にキャストすることができる粘性、均一な溶液です。<sup&gt; [1、4、5]</sup&gt;。私たちの組織構造の場合、我々は標準的な手順を用いてヒツジ胎仔の皮膚から単離された1型コラーゲンを採用<sup&gt; [6]</sup&gt;。いったん組織は37強化しています°コンストラクトが水没するまでC、文化メディアは慎重にプレートに追加されます。設計組織は、それがために準備が整った時点で、さらに2日間の脱水によってさらに凝縮して許可されています<em&gt; in vitroで</em&gt;評価または外科的移植。

Watch this Scientific Journal Video about Fabrication of Myogenic Engineered Tissue Constructs at JoVE.com

Fabrication of Myogenic Engineered Tissue Constructs

ここで、我々は、骨格筋芽細胞を含むコラーゲンベースの、組織構造の作製を実証する。これらの3次元設計された構造は、組織を交換または修復するために使用されることがあります<em&gt; in vivoで</em&gt;。我々の目的のために、我々は、完全心ブロックの修復のための房室の電気コンジットとしてこれらを設計している<sup&gt; [1]</sup&gt;。

筋原培養組織コンストラクトの作製

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

fabrication-of-myogenic-engineered-tissue-constructs

Research

JoVE Journal

Biology

10.4K ビュー.  Children's Hospital Boston and Harvard Medical School. ここで、我々は、骨格筋芽細胞を含むコラーゲンベースの、組織構造の作製を実証する。これらの3次元設計された構造は、組織を交換または修復するために使用されることがあります in vivoで。我々の目的のために、我々は、完全心ブロックの修復のための房室の電気コンジットとしてこれらを設計している [1]。

ビデオ: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

PDMSデバイスの作製と表面改質

生物学

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

熱可塑性マイクロ流体チャネルの作製

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

アンペロメトリー電極の作製

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

ラット心臓における培養組織の移植

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

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 ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

における相同組換えコンストラクトを遺伝子改変<em&gt;ショウジョウバエ</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

基礎脊椎動物の系統からの一次筋原前駆細胞/筋芽細胞培養液の調製

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

ターゲティングされたのsRNAのファージ媒介送達はにおける遺伝子発現をノックダウンするために構築します<em&gt; E。大腸菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

生後マウスにおける聴覚ブラの解剖：中耳の骨の単離および組織学的解析

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...