試薬とマウスのWeiguo Zou教授と加藤先生、そしてZou研究室のメンバーに有益な議論を感謝します。また、上海第9人民病院頭蓋顔面異常研究センターのデジタル化された病頭学研究センターに対する支援に感謝します。この研究は、中国国立自然科学財団(NSFC)からの助成金によって部分的に支えられました[81570950、81870740、81800949]、 上海サミット・高原分野、上海精密医学研究所、上海九人民病院、上海交通大学医学部(JC201809)、上海交通大学医学部の高レベルイノベーションチームインセンティブプロジェクト上海交通大学医学部上海交通大学附属病院(JYJC201902)の学際研究基金L.J.は、優れた青少年医療人材、上海「医療人材の新星」青少年育成プログラム、上海交通大学の「陳興」プロジェクトの学者です。

ここに記載されている動物に関するすべての方法は、上海交通大学医学部の施設動物のケアと使用委員会(IACUC)によって承認されました。
1. 破骨細胞特異的 Stat3 欠失マウスの繁殖
注 :Stat3fl/fl マウスは商業的に得られた。 Ctsk-Cre マウスは、加藤(東京大学、東京12)によって提供された。マウスは、標準化された条件下で施設内の動物施設で特異的な病原体を含まない(SPF)状態下で飼育および維持した。
<ol><li>性的に成熟した雄のマウスと同じ年齢の2匹の雌マウスを組み合わせる。18日後、新生児の毎日のチェックをチェックしてください。妊娠中の雌マウスを分離し、必要に応じて単独で保管してください。雌マウスがペアリングから1ヶ月以内に妊娠しなかった場合、異なる繁殖ケージの間で雄マウスを変更する。</li>
	<li>Ctsk-Creマウス(F0)にStat3fl/flマウスを渡る。ジェノタイピングのための尾をクリップし、男性のStat3flを保つ/+;Ctsk-Creマウスは、生後約6週間である性的に成熟するまでである(F1)。次のプライマーを使用してください: 統計 3 F-TTGACCTGTGCTCC TACAAAAAAAAAAA;統計 3 R-CCCTAGATTAGGCAGCACA;クツク・クレ F-GAACGCACTGcAtTCGACCA;クツク・クレ R-GCTAACCGC GTTTTCGTTTTCC.</li>
	<li>クロス 6 週齢 男性Stat3fl/+;メスのStat3fl/flマウスを持つCtsk-Creマウス。ジェノタイピングのための尾をクリップし、男性Stat3 fl / flを保ちます。Ctsk-Creマウスは、生後約6週間である性的に成熟するまでである(F2)。</li>
	<li>クロス 6 週齢 男性Stat3 fl/fl;メスのStat3fl/flマウスを有するCtsk-Creマウス(F3)。ジェノタイピングのために尾をクリップし、古い繁殖マウスを時間内に若いマウスに置き換えます(F3+N)。</li>
</ol>2. 標本収集
<ol><li>8週齢の雄 Stat3fl/fl と Stat3Ctsk littermateマウスの6組を二酸化炭素窒息で別々に安楽死させる。 注:CO2 の流量は、1分間のケージ体積の30%を置き換えます(例えば、45 cm x 30 cm x 30 cmケージの場合、CO2 の流量は40 L/minです)。</li>
	<li>マウスをスピーヌの位置に配置します。両側股関節を手で優しく外す。眼科用はさみを使用して遠位脛球から皮膚を垂直に切り落とし、後肢から皮膚全体を解剖する。</li>
	<li>右股関節と膝関節の関節靭帯をハサミで切り取り、後肢を分離する。トロシャンターとフィブラの接合部を切り、後肢を4%パラホルムアルデヒドに浸します。ステップ 3 の右後肢を保持します。適切に完全に浸漬し、4%パラホルムアルデヒドで骨髄を固定するために両端の骨をカット.</li>
	<li>左股関節と膝関節の関節靭帯をはさみで切断し、柔らかい組織をそっと取り除き、脛骨と大腿骨を慎重に分離します。脛骨と大腿骨を75%エタノールに分けて浸します。ステップ4のフェムラとステップ6の脛骨を保ちます。トロチャンターをそのままに保つようにしてください。</li>
</ol>3. パラフィンセクションの準備
<ol><li>4°Cで4%パラホルムアルデヒドで48時間右後肢を固定します。</li>
	<li>脱灰: 1x PBSで10分間3回静かに洗浄します。骨が曲がることができるまで3〜4週間超音波デカーシファイアで15%EDTA(ddH2Oの800 mLおよび100 mLの100 mLで150 g EDTA)の試験体を脱灰する。1日おきに新鮮な脱カルシウム液に交換してください。</li>
	<li>試料を1x PBSで3倍軽く洗い、4°Cで75%エタノールに浸します。</li>
	<li>脱水:2日目に、95%エタノール、100%エタノール、キシレンに検体を順次浸漬し、それぞれ1時間ずつ2回浸します。</li>
	<li>1/2キシレン1/2パラフィンに30分間試料を浸します。試料をパラフィンに65°Cで一晩浸漬します。</li>
	<li>埋め込み: 埋め込み用の適切な埋め込みタンクを選択します。脛目を下に均一に置きます。大腿骨と脛骨を90°の角度で配置します。パラフィンを完全に冷却し固めた後、埋め込みタンクから取り出します。標本に番号を付け、一晩で-20 °Cで保管してください。</li>
	<li>マイクロトームを使用して、厚さ5μmの部分を連続的にカットします。20~40のセクションをカットします。37°Cの水にセクションを広げ、顕微鏡スライドに付着し、一晩42°Cで焼きます。</li>
</ol>4. マイクロCTスキャンおよび分析
<ol><li>マイクロCTスキャナで左フェムラをスキャンします。解像度:10 μm。電圧:70キロV。電流:114 μA;フリッター:0.5ミリメートルアル;回転ステップ:0.5°。</li>
	<li>メーカーの指示に従ってスキャナーのサポートソフトウェアを使用して、皮質骨と骨の3D画像を再構築します。ROIは、遠位成長プレートに近い骨の合計1mm幅、および女性の真ん中の皮質骨の合計1mm幅の幅である。</li>
	<li>定量的マイクロアーキテクチャパラメータを計算します:骨密度(BMD)、骨体積分率(BV /TV)、骨膜厚(Tb.Th)、柱状数(Tb.N.)、骨膜分離(Tb.Sp.)、皮質骨厚さ(Ct.Th)。</li>
</ol>5. トラップ染色
<ol><li>パラフィンの切片を65°Cで30分間焼きます。</li>
	<li>デワックス:10分間、切片をキシレンに浸します。このステップ3xを新鮮なキシレンで実行します。</li>
	<li>リハイドレート:セクションを100%エタノール、95%エタノール、70%エタノール、およびddH2Oに順次浸漬し、それぞれ5分間2回浸漬します。</li>
	<li>製造者の指示に従ってTRAP染色キットを使用して染色液を準備し、37°Cに温めます。 注:TRAP染色溶液は、すべてのアッセイの直前に新たに調製する必要があります。</li>
	<li>50~100 μLの染色液を各サンプルに加え、37°Cの湿気のあるチャンバーで20~30分間インキュベートします。赤い多核破骨細胞が見えるまで、5分ごとに軽い顕微鏡で破骨細胞の染色状態を確認してください。反応をddH2Oで終了する。</li>
	<li>30 sのためのヘマトキシリン溶液中のカウンターステイン。1%アンモニア溶液に1分間浸漬することで、安定した青色を作成します。ゆっくりと水道水を流してすすい。</li>
	<li>ニュートラルバルサムを使用してカバースリップでセクションをマウントし、一晩乾燥させます。</li>
	<li>顕微鏡で関心のある3~5の分野を捉えます。イメージ J で境界の分析: '直線' ツールを使用してスケール バー (Ls) の長さを測定し、L2、物理長 (Lp)= Ls*L2/L1) として 'セグメント線' ツールを使用して、境界の長さを測定します。3つ以上の核を持つTRAP陽性細胞の数を数えます。</li>
</ol>6. カルセインとアリザリンレッドダブルラベリング
<ol><li>標本調製:腹腔内注入 20 mg/kg カルセイン (2% NaHCO 3溶液で 1 mgL 2% NaHCO3 溶液) 4日目に 25 mg/kg アリザリンレッドS (AL,H 2 Oで 2 mg/mL) 4日目.7日目にマウスを犠牲にする。48時間4°Cで4%パラホルムアルデヒドに慎重に関連付けを解除します。</li>
	<li>脱水: 固定後、1x PBSで脛目3xを静かに洗います。検体を95%エタノール、100%エタノール、キシレンを5分間、別々に2回浸漬します。</li>
	<li>試料をアセトンに12時間、1/2アセトン1/2樹脂に2時間浸し、純粋な樹脂を一晩乾燥オーブンに浸します。 注:樹脂は、メーカーの指示に従って市販の樹脂(例えば、埋め込み812樹脂)によって調製した。</li>
	<li>埋め込み: 純粋な樹脂を適切なシリカゲル埋め込みタンクに加え、泡を避けるために試料を穏やかに配置します。乾燥オーブンで樹脂を60°Cで48時間重合します。</li>
	<li>回転ミクロトームで試料を5μmの厚いセクションに連続して切ります。残りのサンプルは、乾燥剤とともに室温で保管してください。</li>
	<li>75%のアルコールの低下でピンセットでセクションを付けます。ニュートラルバルサムを使用してカバーリップでセクションをマウントします。蛍光顕微鏡で赤と緑の蛍光標識をキャプチャします。</li>
	<li>2 つのラベル線(Ir.L.Wi)、単一ラベル付きの柱状周長(sL.Pm)、二重ラベル付きの柱状の周長(dL.Pm)、および総柱状周長(Tb.Pm)の間の幅を測定します。鉱物割当率(MAR)と骨形成率(BFR/BS)を計算します。MAR = Ir.L.Wi/インターバル日数。BFR/BS= (dL.Pm±SL.Pm/2)*MAR/Tb.Pm*100%。</li>
</ol>

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著者らは開示するものは何もない。

遺伝子組み換えマウスモデルは、ヒト疾患13のメカニズムおよび医薬的治療の研究に一般的に使用される。 Ctsk-Cre マウスは、破骨細胞6の機能研究に広く使用されている。本研究では、骨格表現型を分析し、生体内の破骨細胞活性を制御する重要な遺伝子を研究する方法のプロトコルについて説明した。
組織学的分析は骨代謝を?...

骨格骨は人体の主な荷重を受ける器官であり、歩行と運動1の間に内部環境と外部環境の両方から圧力を受けている。骨は骨芽細胞と破骨細胞のバランスが取れた自己再生を通り抜け続ける。破骨細胞が古い骨を除去し、骨芽細胞が新しい骨を形成する過程は、骨格系2の恒常性および機械的機能を維持する。バランスの乱れは骨粗鬆症などの骨代謝性疾患を誘発しうる。骨粗鬆症は、過剰な破骨活性によって引き起こされ、世界的に流行しており、社会2、3、4に大きな経済的損失を引き起こす。骨粗鬆症治療に利用できる限られた数の薬物と副作用のリスク4によると、骨細胞形成および活動の詳細を明らかにすることが重要である。
単球/マクロファージ造血系統に由来する破骨細胞は、複数の核(2〜50核を有し得る)を有し、大きい(通常、直径100μmを超える)2である。骨破骨疾患のメカニズムの探索と薬剤のスクリーニングは、体外骨細胞内培養を通じて広く改善されているが、複雑な有機反応は、標的療法に不可欠なインビボの証拠を作る。マウスとヒトの間の遺伝的および病的生理学的類似性のために、遺伝子組み換えマウスモデルは、生体内でのヒト疾患のメカニズムおよび医薬的治療法を研究するために一般的に使用される。Cre-loxPシステムは、マウス遺伝子編集に広く使用されている技術であり、研究者は組織/細胞特異的な方法で遺伝子機能を調査することを可能にしました 5.カテプシンK(CSTK)は、骨コラーゲン8を分解し得る破骨細胞によって分泌されるシステインプロテアーゼである。CTSKは成熟した破骨細胞で選択的に発現することは十分に受け入れられています。従って、Ctsk-Creマウスは破骨細胞の機能研究に有用なツールであると考えられ、6.
転写(STAT)ファミリーのシグナルトランスデューサおよびアクチベーターは、免疫と癌の進行と発達において古典的かつ非常に重要です7,8.7つのSTAの中で、STAT3は骨恒常性9,10に最も関連していると報告されている。いくつかのインビボ研究は、骨芽細胞におけるSTAT3の特異的不活性化が骨形成9、10を減少させることを報告している。それにもかかわらず、骨細胞形成および生体内の骨代謝におけるSTAT3の関与に関する確かな証拠は依然として限られている。最近、我々は、骨細胞特異的Stat3欠失マウスモデル(Stat3fl /fl;Ctsk-Creは、STAT3と呼ばれるSTAT3)は、STAT3が破骨細胞分化および骨代謝に関与することを11.本研究では、骨骨格特異的STAT3欠失が骨恒常症に及ぼす影響を研究するために、Stat3Ctskマウスの骨量、骨組織形態、骨代謝および異化の変化を分析するために用いた方法とプロトコルについて説明する。

<table>
	<tbody>
		<tr>
			<td>4% Paraformaldehyde solution</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E672002</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>80000360</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia solution</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-Cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>Electron Microscopy Sciences</td>
			<td>13710</td>
			<td></td>
		</tr>
		<tr>
			<td>DeCa RapidlyDecalcifier</td>
			<td>Pro-Cure</td>
			<td>DX1100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>Electron Microscopy Sciences</td>
			<td>13600</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBED 812 RESIN</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Beyotime Biotechanology</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT</td>
			<td>Scanco Medical AG</td>
			<td>&#956;CT 80</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>10018918</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E675007</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>Electron Microscopy Sciences</td>
			<td>19000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>A601889</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary microtome</td>
			<td>Leica</td>
			<td>RM2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Stat3fl/fl mice</td>
			<td>GemPharmatech Co., Ltd</td>
			<td>D000527</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

skeletal phenotype analysis of a conditional stat3 deletion mouse model

トランスジェニックマウスモデルは、破骨細胞の分化と活性を制御する重要な遺伝子を理解し、骨粗鬆症のメカニズムと薬学的治療法を研究するのに強力です。カテプシンK(Ctsk)-クレマウスは、破骨細胞の機能研究に広く使用されています。転写3(STAT3)のシグナルトランスデューサおよび活性化剤は骨恒常性に関連しているが、生体内の破骨細胞におけるその役割は未定のままである。STAT3が破骨細胞分化と骨代謝に関与しているというインビボの証拠を提供するために、我々は、破骨細胞特異的Stat3欠失マウスモデル(Stat3 fl/fl;Ctsk-Cre)を、骨格表現型を分析した。マイクロCTスキャンと3D再構成は、条件付きノックアウトマウスにおける骨量の増加を暗示した。H&E染色、カルセインおよびアリザリンレッド二重染色、および酒石酸抵抗性酸ホスファターゼ(TRAP)染色を行い、骨代謝を検出した。つまり、このプロトコルは、骨格表現型を分析し、生体内の破骨細胞活性を制御する重要な遺伝子を研究するためのいくつかの正規の方法と技術を記述する。

本プロトコルを用いて、破骨細胞特異的Stat3欠失マウスを生成し、破骨細胞分化に対するSTAT3欠失の影響を検討した。Stat3Ctskマウスとその野生型(WT)リターメイトは、ジェノタイピング後に飼育され、保管された。骨髄マクロファージを単離し、破骨細胞に培養し、Stat3CtskマウスにおけるSTAT3欠失が実証された(図1)。
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Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

このプロトコルは、生体内の破骨細胞活性を制御する重要な遺伝子を理解するための正規的方法を記述する。この方法は、トランスジェニックマウスモデルといくつかの正規の手法を用いて骨格表現型を分析する。

条件付き Stat3 欠損マウスモデルの骨格表現型解析

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

skeletal-phenotype-analysis-conditional-stat3-deletion-mouse

Research

JoVE Journal

Biology

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

4.3K ビュー.  National Clinical Research center of Stomatology. このプロトコルは、生体内の破骨細胞活性を制御する重要な遺伝子を理解するための正規的方法を記述する。この方法は、トランスジェニックマウスモデルといくつかの正規の手法を用いて骨格表現型を分析する。

ビデオ: Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

出芽酵母のタンパク質濃度の関数としての形態学的表現型の開発の分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

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 ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

複屈折およびタッチ呼び起こすエスケープ応答アッセイによる幼虫のゼブラフィッシュ骨格筋の欠陥の分析

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

ヒト多能性のための代替培養はセル生産、メンテナンス、および遺伝子解析幹

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

マウス骨格筋におけるDNA損傷の検出と分析<em&gt;その場で</em&gt; TUNEL法を用いた

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

遺伝子発現と組織学的分析のためにシングルマウス骨格筋の連続領域の凍結切片化

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

腰椎不安定性のマウスモデル

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...