カーン博士は、NIH/NICHD K12HD052892、アルファ 1 の基礎、ヒルマン財団およびピッツバーグ肝臓研究センターから助成を認めています。博士 Michalopoulos は、NIH/NIDDK P01DK096990 から助成を認めています。アン ・ オアの優秀で、寛大な技術的な支援お願い申し上げます。マイクロサージャリー移植または齧歯動物ラボへのアクセスを提供するためも博士デビッド A. ゲラーに感謝しています。

動物介護制度とピッツバーグ大学の利用委員会の倫理的なガイドラインに従ってすべてのプロシージャを行った。
1. 基本的なテクニックと一般的な手順
<ol><li>オートクレーブ使用する前にすべての顕微鏡装置。</li>
	<li>麻酔によりマウスを殺すことを避けるため、慎重にその呼吸パターンを確認します。</li>
	<li>臓器や器官を損傷し、出血を引き起こしているを避けるために可能な限り少し腸をタップします。湿式の綿棒やガーゼで出血点を押してください。</li>
	<li>臓器の穏やかな操作にウェット綿棒を使用します。2 つのサイズでウェット、非織ガーゼを使用、3.5 × 3.5 cm と 6.0 × 6.0 cm。 は全肝を撤回する小さいものを使用します。大きい 1 つを使用して、小規模および大規模な腸をラップします。</li>
</ol>2. 術前準備
<ol><li>男性の c57/b6 マウス部分胆管結紮用 (体重が約 20 35 g) 時代の 8 〜 12 週間を使用します。</li>
	<li>麻酔導入までマウス水や食料への無料アクセスを許可します。</li>
</ol>3. 部分胆管結紮操作
<ol><li>1.5 から 2.0 パーセントのイソフルランと 1 L/分の酸素を混ぜた誘導の商工会議所に動物を置くことによってマウスを麻酔します。呼吸を観察することによって動物を注意深く監視します。動物の呼吸速度は 1 秒あたり 1 つの息をマウスがよく麻酔です。以来、前肢逃避反射はより急速に消え、逃避反射の完全な不在を確認する同様に後肢の足をピンチします。目は、角膜潰瘍を防ぐために眼科用潤滑剤と潤滑する必要があります。メモその鎮痛剤の投与は痛みの受容体の刺激を防ぐために手術前に与えることができます。</li>
	<li>シェイブ バリカン、腹壁、手術用顕微鏡下で手術のテーブルの上にマウスを置きます。テーブルにすべての手足をテープで固定します。イソフルラン吸入を全身麻酔下でマウスを維持するために使用します。<ol>
<li>イソフルラン気化器を調整し、誘導 2.0 3.0% イソフルランを使用します。</li>
	</ol>
</li>
	<li>腹部壁の消毒滅菌綿棒を使用して、最初 0.3 0.5 mL ポビドン ヨードの局所的に適用は 70% のエタノールが続きます。動物は、滅菌ドレープで覆われるべきであります。</li>
	<li>はさみとアドソン鑷子を使用して恥骨に、剣から正中腹部切開を確認します。0.8 1.0 にイソフルランを減らす腹部切開をしたら、メンテナンスのため %。</li>
	<li>マイクロ リトラクターを使用して腹腔内を公開します。モスキート鉗子で、剣を押し、マウスの頭に向かって撤回します。ソフト粘土を使用して場所のモスキート鉗子を修正します。</li>
	<li>温めた (37 ° C) の 1-2 の mL を植え付ける滅菌リン酸緩衝生理食塩水 (PBS) 腹部臓器が負傷を避けるために腹腔内に。濡れた綿棒で小腸を押し軽くマウスの頭に向かってそれらを撤回します。小腸、小腸 (図 1 a) を負傷を避けるために後腹膜間靭帯をカットします。</li>
	<li>ウェット、不織布-ガーゼ スポンジでの小規模および大規模な腸をラップします。腹腔内の尾側に配置します。滅菌ガーゼに包まれた腸は、汚染を避けるため無菌ドレープ フィールドを配置できます。</li>
	<li>優しく肝門部、肝十二指腸靭帯 (図 1 b) を公開する小型湿式不織布-ガーゼ スポンジを使用して、剣に向かって全体の肝臓を撤回します。</li>
	<li>肝門部の解剖を確認を確認します。総胆管は門脈の腹側にあります。肝門部の主な胆道合流地点に流出する右および左の肝の胆管の位置を確認します。</li>
	<li>左葉を排水左胆管を識別し、縫合針 (図 2 a) の鈍側を使用して 10-0 ナイロン縫合糸とそれを取り囲みます。避けるために門脈、肝動脈、肝表面を負傷します。</li>
	<li>10-0 ナイロン左胆管を結紮します。(図 2 b)。</li>
	<li>湿式の綿棒を使用して、操作の前と同じ位置で小腸・大腸を配置します。</li>
	<li>腸管の捻転と腹腔内出血がないことを確認し、筋肉と 4-0 vicryl の二層で皮膚を閉じます。皮膚縫合用編組縫合 (Vicryl) が使用されたが、感染の可能性を減らすためにモノフィラメントを優先します。 注: は、恥骨に剣から継続的な縫合糸で筋膜を閉じて剣に恥骨から継続的な縫合で皮膚を閉じます。</li>
	<li>切開部を閉じる後麻酔を停止します。</li>
</ol>4. 術後の治療とフォロー アップ
<ol><li>ちょうど手術後がマウスの背側の皮をつまむし、注入 Buprenex (0.1 mg/kg) とセファゾリン (100 mg/kg 皮下、それぞれ Buprenex 注入による 12 時間間隔 3 日、セファゾリンのすべての 24 h 3 日間続いた11,12 ,13。メモその鎮痛剤の投与は痛みの受容体の刺激を防ぐために手術前に与えることができます。セファゾリンと他の周術期の抗生物質だけ移植実験で使われたもののような免疫抑制動物に必要なです。 注: 場合手術を正常に実行すると、マウス通常回復 30 分以内、マウスの活動は術前の状態とほぼ同じになります。</li>
	<li>毎日苦痛の兆候のマウスを確認してください。<ol>
<li>PBDL 後肝臓と (すなわち7、14、21 日) 定義された時点で他の組織サンプルを調達する全身麻酔下頚部転位によってマウスを犠牲に。</li>
	</ol>
</li>
</ol>

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著者の開示があります。

CBDL は閉塞性胆汁うっ滞のため最も一般的な実験的手法が、齧歯動物の複数の課題が発生することができます。術後日のエンドポイントを達成するために必要な数、に応じて、動物は重要な罹患率と時間が進むにつれて死亡率を体験できます。肝臓病の異常肝実質とベースラインで関数を示すいくつかのトランスジェニック マウス モデルを操作するとき、組織の損傷および胆管壊死の程度が...

急性肝障害・肝部分切除や胆管結紮 (cBDL) などの修復手術のモデルは、肝再生と病理学1,2を研究する齧歯動物で何十年も使用されています。CBDL で (これは肝臓で生成されるすべての胆汁を排出) 胆管結紮は、肝硬変3に最終的な進行すると術後最初の 2 〜 3 週間で閉塞性胆汁うっ滞、炎症および線維化の結果します。胆管の増殖、分化転換、および居住者の肝前駆細胞の誘導 cBDL4、5の機能があります。CBDL は、再現可能な成果野生型マウスと正常な肝臓6,7,8でこれらの系統の特徴である閉塞性 cholangiopathy の特定のメカニズムを解明し、プロシージャは、肝疾患、高い罹患率と死亡率は時間の経過とともに胆道閉塞のため予想よりもより多くの動物を必要とするいくつかの遺伝子導入マウスモデルで技術的に厳しいことができます。この手法も長時間コース実験 cBDL のストレスをそれ以外の場合は許せないかもしれないトランスジェニック マウスの基になると肝疾患の勉強のために有益です。
pBDL は、いくつかのこれらの課題を克服するために合理的な選択肢を提供できます。野生型マウスの初期 pBDL 研究は、炎症9の演技のローカルおよび全身の仲介人を区別するために目指しています。PBDL、総胆管の上左右の肝胆汁管の主な胆道合流は慎重に、門で可視化し、ローカライズされた閉塞性胆汁うっ滞を生成するのみ、左肝内胆管を結紮します。pBDL では、cBDL にいくつかの利点を提供しています。PBDL、unligated の右葉は栄養状態や要因を循環など、同じ全身作用を受ける結紮の左葉の同じ牌内部統制として機能します。全身性炎症性メディエーターを調べたところ、大沢ら以下の線維化および unligated の葉の壊死が観察が、炎症細胞と変形の成長因子 β 式結紮葉9に増加します。
最近では、pBDL が保持されて胆汁10の肝実質の効果を研究する再定義されました。(Unligated「シャム」) 内部統制群と実験群の同じ動物を使用して、pBDL は効果的により費用対効果は実験ごとに必要な動物の数を半減します。マウスは、コース実験理想的な時間 (≥2 週) 結紮葉で達成することができますので、はるかに良い、pBDL の影響を容認します。最も重要なは、pBDL は閉塞性胆汁うっ滞の直接的な肝内効果を区別することができます、unligated コントロール葉に比べて、結紮葉の胆汁構成成分を保持します。全体的に、pBDL は肝臓でローカライズされた胆汁うっ滞の葉固有の効果を研究する魅力的な方法です。

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			<td height="21" style="height:21px;width:343px;">Microscope-Leica Wild M650, 6&#8211;40&#215; magnification</td>
			<td style="width:336px;">Leica Microsystems</td>
			<td style="width:195px;">n/a</td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="21" style="height:21px;">Tec 3 isoflurane funnel fill vaporizer</td>
			<td>General Anesthetic Services</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stevens Tenotomy Scissors</td>
			<td>Accurate Surgical &#38; Scientific Instruments Corporation</td>
			<td>ASSI.9193</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td>Fine science tools</td>
			<td>11006-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mosquito classic delicate hemostatic forceps</td>
			<td>Codman</td>
			<td>30-4472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-retractor</td>
			<td>Murdock; Roboz Surgical Instrument</td>
			<td>RS-6550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonwoven gauze sponges</td>
			<td>Fisherband</td>
			<td>870-PC-DBL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puritan 6&#8221; Small Cotton Swab w/Wooden Handle</td>
			<td>Puritan Medical Products Company</td>
			<td>868-WCS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont SS Forceps - Standard Tips/Straight/Inox/13.5cm</td>
			<td>Fine science tools</td>
			<td>11203-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont SS Forceps - Standard Tips/Angled 45&#176;/Inox/13.5cm</td>
			<td>Fine science tools</td>
			<td>11203-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine science tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microneedle holder</td>
			<td>Aesculap Surgical Instruments</td>
			<td>FD231R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro AROSuture, sterile 10-0 nylon suture, 70 &#181;m, TAP points</td>
			<td>AROSurgical Instruments Corporation</td>
			<td>T4A10N07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl</td>
			<td>Ethicon</td>
			<td>J662H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-ml Syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon tissue culture dish, 60 &#215; 15 mm</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:343px;">Isoflurane, United States Pharmacopeia (USP) liquid for inhalation, 250 ml</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenex injectable (buprenorphine hydrochloride)</td>
			<td>Reckitt Benckiser Pharmaceuticals</td>
			<td>NDC 12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefazolin for injection , USP</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-238-61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVP scrub solution (povidone&#8211;iodine 7.5%)</td>
			<td>Medline</td>
			<td>NDC 12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Phosphate Buffered Saline Without Calcium or Magnesium</td>
			<td style="width:336px;">LONZA</td>
			<td style="width:195px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sirius Red</td>
			<td style="width:336px;">Sigma</td>
			<td style="width:195px;">365548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Hematolxylin</td>
			<td style="width:336px;">Fisher (Richard-Allan Scientific)</td>
			<td style="width:195px;">22-050-111 (7211)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Eosin</td>
			<td style="width:336px;">Anatech (Fisher)</td>
			<td style="width:195px;">832 (NC9686037)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Formalin</td>
			<td style="width:336px;">Fisher</td>
			<td style="width:195px;">SF100-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

partial bile duct ligation in the mouse: a controlled model of localized obstructive cholestasis

総胆管の完全な胆管結紮 (cBDL) は、齧歯動物、閉塞性胆汁うっ滞、肝内胆管の増殖を研究するための確立された手術方法です。ただし、長期的な実験は増加罹患率と死亡率につながることができます。選択マウス系統基礎疾患、単一動物の損失および費用を減らすことができます肝葉の結紮とも有意義な比較を可能です。ここで、説明部分胆管結紮 (pBDL) にのみ左肝内胆管を結紮は、マウスの左葉ですがない、残りの葉の胆管閉塞の原因します。同じ動物で同じ条件に服従するときに unligated の葉は、結紮葉に内部統制として役割を果たすため注意して顕微鏡手術と pBDL 実験が費用対効果をすることができます。CBDL とは異なり、別の偽手術群は必要ではありません。pBDL、胆汁うっ滞及び他の保有胆汁成分の全身影響とローカライズされた直接の比較に非常に役立ちます。pBDL は薬や細胞遊走に関連するメカニズムを調査する手法として用途に使用することができます。

この典型的な実験のため pBDL は、α 1 アンチトリプシン (A1AT) 欠乏は、人間の肝臓病を要約する帳票のピッツ ・ トランスジェニック マウスで行われました。PiZ マウス染色遺伝子の DNA の突然変異人間 A1AT 遺伝子、プロモーター、イントロンと上流と下流地域14の側面の ~ 2 kilobases のコーディング領域を含む複数のゲノム断片から成る <sup class...

Watch this Scientific Journal Video about Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis at JoVE.com

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

肝臓の傷害および齧歯動物の再生の手術モデルとして部分胆管結紮を紹介します。

マウスの部分胆管結紮: ローカライズされた閉塞性胆汁うっ滞の制御モデル

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

partial-bile-duct-ligation-mouse-controlled-model-localized

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.4K ビュー.  Jichi Medical University. 肝臓の傷害および齧歯動物の再生の手術モデルとして部分胆管結紮を紹介します。

ビデオ: Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

マイクロ流体プローブ：局在表面処理のための操作と使用方法

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

生物学

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

LAD -ライゲーション：心筋梗塞のマウスモデル

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

内リンパ管の閉塞による内リンパ水腫の外科的誘導

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

マウス乳腺に局在薬物送達のための管内注入

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

細胞特異的にLINC複合体を破壊するマウスモデルの検証

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

腸の部分的な閉塞のマウス モデル

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

インビトロにおける胆道カネラ形成における細胞骨格の役割を研究する原発マウス肝細胞の単離と3Dコラーゲンサンドイッチ培養

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

マウスの部分胆管結紮: ローカライズされた閉塞性胆汁うっ滞の制御モデル

要約

さらに動画を探す

この動画の章

マイクロ流体プローブ：局在表面処理のための操作と使用方法

LAD -ライゲーション：心筋梗塞のマウスモデル

内リンパ管の閉塞による内リンパ水腫の外科的誘導

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

マウス乳腺に局在薬物送達のための管内注入

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

細胞特異的にLINC複合体を破壊するマウスモデルの検証

腸の部分的な閉塞のマウスモデル

インビトロにおける胆道カネラ形成における細胞骨格の役割を研究する原発マウス肝細胞の単離と3Dコラーゲンサンドイッチ培養

マウスの部分胆管結紮: ローカライズされた閉塞性胆汁うっ滞の制御モデル

要約

さらに動画を探す

この動画の章

マイクロ流体プローブ：局在表面処理のための操作と使用方法

LAD -ライゲーション：心筋梗塞のマウスモデル

内リンパ管の閉塞による内リンパ水腫の外科的誘導

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

マウス乳腺に局在薬物送達のための管内注入

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

細胞特異的にLINC複合体を破壊するマウスモデルの検証

腸の部分的な閉塞のマウス モデル

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