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総合的なプロトコルには、敗血症の誘導の最初の日と、動物とマウス BALF からエクソソーム隔離のため 2 日目から BAL 液の分離を含む 2 日が必要です。すべての手順が検討されており、機関動物ケアおよびアトランタ VA メディカル センターで使用委員会によって承認されました。
1. マウス急性敗血症性肺傷害モデル
<ol><li>6-8 週間-を使用して古い男性野生型 c57bl/6 j マウス (重量 20 22 g) 動物のモデル、および無菌技術と機器を使用してすべての手順を実行します。オートクレーブすべて手術器具、鉗子、はさみ、121 ° C で 20 分用を含む</li>
	<li>実験的にマウスとコントロール グループをランダムに配布します。手術用ボード上でマウスを抑制します。腹腔内のルートによる大腸菌リポポリサッカライド (LPS、15 mg/kg) を持つマウスを扱います。個人体重別に適切な LPS 投与量は 0.1 mL PBS マウスあたりに希釈し、27 G の針を用いて 1 mL 注射器を使用して LPS 溶液を注入して、対照動物に PBS の相当量を注入します。</li>
	<li>適切な室温できれいなケージから成っている動物の BSL2 施設にマウスをしてください。</li>
	<li>24 h 後、CO2吸入マウスを安楽死させます。</li>
	<li>マウスの首のセンターで外科の領域を選択します。(約 5 mm) の小切開を行い、気管、気管を露出するまで鈍い鉗子を使用してへのアクセスのための筋肉を優しきます。</li>
	<li>27 G 針付きマウス BAL 液 10 mL 滅菌注射器とを撤回します。まず、優しくと気管に針を挿入、気管に 1 mL の滅菌 PBS をゆっくりと注入し、27 G 針付き 3 mL シリンジに BALF を描画します。プロシージャが完了したら、注射器や針バイオハザード シャープス コンテナーの処分します。</li>
	<li>Chauretらによって前述のように塩素、二酸化炭素、手術器具を扱う27、121 ° C で 20 分間のオートクレーブによるすべての手術器具を滅菌、</li>
</ol>2. エクソソーム分離シリアル超遠心分離とキャラクタリゼーション
<ol><li>15 mL の円錐管にマウス BAL 液を収集します。肺胞細胞を収穫するには、遠心分離機の 4 ° C での 1,000 g で 10 分間のサンプル</li>
	<li>上清を新しい 15 mL コニカル チューブに転送し、4,000 x g 細胞の残骸を削除する 4 ° C で 30 分間試料を遠心分離します。</li>
	<li>超遠心機チューブと大きい細胞の粒子を除去する 4 ° C で 10,000 x g で 60 分間遠心分離機サンプルの上澄みを収集します。</li>
	<li>上清を新しい円錐管に転送し、試料の残りのより大きい粒子を削除する 0.2 μ m シリンジ フィルターを通して。</li>
	<li>超遠心機チューブ内のサンプルを収集し、管のバランスをとる。遠心分離機のサンプル、エクソソームのペレットへの 4 ° C で 140,000 x g で 2 時間。 注: エクソソームのサイズは 20-200 nm を異なります。200 よりも大きな粒子を除去するため 0.2 μ m フィルターを使用サイズの nm。</li>
	<li>優しく培養上清を捨てます。100 μ L の PBS または溶解・ ソリューション、1.5 mL チューブに転送エクソソーム サンプルとペレットを溶解し、使用するまで-80 ° C で保存します。 メモ: 分離エクソソームを確認するには、透過型電子顕微鏡の使用をお勧めします。</li>
	<li>処理プロトコル28標準 TEM サンプルに従ってください。エクソソーム サンプルの透過型電子顕微鏡と取るイメージ スケール 200 バーを使用して分析 nm。</li>
</ol>3. miRNA と舞台 miRNA-q-RT-PCR の準備
注: Mirna の発現レベルを分析するには、miRNA q RT-PCR 法の使用をお勧めします。
<ol><li>標準 miRNAs の隔離のプロトコルに従うし、一本鎖逆転写24で浄化された Rna の同量を使用することを確認します。</li>
	<li>BAL 液中 exosomal マイクロ Rna レベルを検出する標準のリアルタイム PCR のプロトコルに従ってください。ミール 155 やミール-146a U6 SnRNA に対する特定のリアルタイム PCR のプライマーを使用します。定量的 PCR 装置と PCR の反作用を実行し、内部統制 U6 snRNA に式の値を正規化します。 注: 各グループは、帳票サンプル24で構成されています。</li>
</ol>4. エクソソームのラベリングと浄化
<ol><li>1.5 mL ポリプロピレン チューブに 100 μ L 懸濁液ソリューション エクソソーム ペレットを中断し、懸濁液溶液 100 μ L を他の管へエクソソームの染料の 0.4 μ L を追加します。</li>
	<li>すぐに色素ソリューションにソリューションのエクソソームを追加し、ピペッティングでそれを徹底的にミックスします。室温で 5 分間のサンプルを維持、光から保護します。</li>
	<li>エキソソーム スピン列を 1.5 mL 溶出チューブに入れてください。エキソソーム スピン列のセンターにソリューションの混合物を転送します。</li>
	<li>室温で 800 x g で 2 分間列をスピンします。</li>
	<li>列を破棄し、使用するまで-20 ° C で溶出サンプルを格納します。</li>
</ol>5. エクソソームを受信者の細胞によって撮影の検証
<ol><li>1 x 10 の4 8 もチェンバー スライド、37 ° c、5% CO2培養細胞で MLE 12/cell の種子します。</li>
	<li>10 μ L の蛍光ラベル エクソソームをセルに追加します。37 ° C で 1 時間インキュベートします。</li>
	<li>文化メディアを削除し、pbs セルを洗う、室温で 10 分間の 1% パラホルムアルデヒドとセルを修正します。</li>
	<li>DAPI をマウント中のスライドをマウントします。</li>
	<li>蛍光顕微鏡でサンプルを分析し、画像、40 倍の倍率がかかります。</li>
</ol>6. エクソソームと受信者の細胞間の相互作用の検証
<ol><li>種子 2 x 105 MLE12/ウェルおよび 5% CO237 ° C で培養細胞を適切な培地を含む 12 ウェル培養プレート。</li>
	<li>MLE12 セルにマウス BAL 液 20 μ g エクソソームを追加し、実験グループごとの複製を設定します。</li>
	<li>24 h はその後、PBS のセルを洗浄し、ウェルに 600 μ L 換散バッファーを追加します。優しくプレートを振るし、ピペットと 1.5 mL チューブで溶解サンプルを収集します。</li>
	<li>標準の総 RNA の隔離のプロトコルおよび cDNA 逆のトランスクリプション プロトコルに従う、一本鎖 cDNA の逆のトランスクリプション29を完了します。</li>
	<li>受信者セル29の mRNA 発現レベルを検出する標準のリアルタイム PCR のプロトコルに従ってください。TNF α、イルに対して特定のリアルタイム PCR のプライマーを使用して-6、および ZO 1。定量的 PCR 装置との PCR の反作用を実行し、GAPDH の内部のコントロールに式の値を正規化します。 注: 各グループは、帳票のサンプルで構成されています。</li>
</ol>

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この作品は、RS に VA メリット レビュー 2I01BX001786 06A1 資金によって支えられました。

病気のマウス ・ モデルは、特定の遺伝子の生理機能を評価し、実験2のコストを削減する通常使用されます。急性敗血症性肺傷害を説明ここで ARDS の人間に見られる炎症性応答を模倣しています。このモデルは分子病態研究、バイオ マーカーの開発を調査し、潜在的な新しい治療法5をテストするものです。
共培養系研究生体内...

ALI および ARDS は重度の低酸素血症の約 100 万人世界中に影響を与える非心原性肺水腫による呼吸不全の生命にかかわる形態毎年1。ARDS の病因には、感染症や吸引やさまざまな間接的な侮辱から肺に直接傷害が含まれています。最後のディケイドが ARDS の病態理解が増す、しかし、ARDS の特定のターゲットを絞った治療法がまだ2,3を開発します。
急性肺傷害のいくつかの動物モデルは人間の研究4,5実験療法を翻訳するためのブリッジを提供する開発されています。一般的に使用されるモデルには、オレイン酸や細菌、LP、ブレオマイシンのローカル インストールが含まれます。他のアプローチには、虚血再灌流、盲腸結紮穿刺、機械的換気誘発ストレッチ損傷、酸素または細菌の LPS5全身投与が含まれます。これらのモデルは臨床的仮説をテストする有用な生物学的システムを提供する、潜在的な治療法の開発。人間 ARDS をシミュレートするには、動物モデルは炎症や肺のバリア機能の欠陥と上皮と内皮細胞を急性障害を再現する必要があります。
エクソソームは分子の内容には、蛋白質、DNA、RNA および脂質が含まれています、直径 20-200 nm の膜小胞であり、分子組成振込組織微小環境で細胞間のコミュニケーションを円滑します。エクソソームは複数の種類の細胞、内皮細胞、上皮細胞、平滑筋細胞、腫瘍細胞などから分泌されるが、人間の体液に存在します。研究を示すエクソソームは感染性と無菌の炎症性疾患時に免疫細胞と間質細胞間クロストークを規制し、生理中に様々 な自然と実験的刺激で規制するその異常なリリースが表示されます。および病理学プロセス6。このような通信ネットワークは肺疾患の病態に重要な役割を果たす可能性があり、病態生理学的進行7、8に影響を与える可能性があります。として 18-22 ヌクレオチド非コード Rna、miRNAs 組織と体液、血漿、血清に存在し、翻訳後修飾レベル9,10mRNA 発現を調節します。
エクソソーム パッケージ miRNAs 複数種類の細胞の分化と機能に影響を与えるし、様々 な病気、ガン、肺疾患、肥満、糖尿病、心血管疾患11を含む関連付けられている過剰なレベル 12,13,14,,1516。受信者の細胞と exosomal の miRNAs の往復への参入は、微小環境17,18の止血を変更する細胞間のコミュニケーションを促進します。急性肺傷害、エクソソーム8を通じて広範な細胞間コミュニケーションの種類の細胞を含む複雑なプロセスです。ミール 155 とミール 146a 一般的な転写調節機構を共有し、炎症反応や免疫寛容19,20に貢献。最近の研究では、両方が exosomal miRNAs の免疫細胞21間往復経由で炎症反応を調節することを示します。しかし、変調エンドトキシンに歯槽の応答に及ぼす exosomal Mirna の分子機構は不明、間違いなく潜在的な臨床的意義と並進含意メリット詳細な調査。
炎症とがんの22,23などの複雑な環境で特定の細胞型の相互作用を定義する共培養モデルを採用されています。これらのプラットフォームは、特に構造と免疫細胞の種類の細胞間クロストークを尋問する代替方法を提供します。
内気管、LPS の噴霧、腹腔内投与または全身投与、実験的肺傷害24,25,26を誘導するために広く使用され上皮と内皮細胞の透過性を誘導するために示されています。欠陥があります。ここで敗血症モデル マウスにおける急性肺傷害を誘発するのに腹腔内の LP を使用します。LPS の腹腔内投与の 24 時間以内の炎症細胞と肺の透過性欠陥が誘導されます。さらに、バルからエクソソーム含ま miRNA 155 とミール-146a と BAL 液からエクソソームは il-6, TNF-α を含む受信者の上皮細胞で炎症性サイトカイン発現を誘導することを示す.これらのデータは、その exosomal の Mirna を示す第 1、急性敗血症性肺傷害のこのモデルのバルで分泌されるが。

<table border="0" cellpadding="0" cellspacing="0" style="width:1276px;" width="1276">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:691px;">lipopolysaccharide</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:224px;">Escherichia coli 055:B5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS pH7.2(1X)</td>
			<td>Life technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit</td>
			<td>Thermo Fisher</td>
			<td align="right">449774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>Thermo Fisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mmu-miR-155 (002571) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">has-miR-146a (000468) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U6snRNA primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF-&#945;(Mm00443258_m1 ) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IL-6 (Mm00446190_m1&#160;&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZO-1 (Mm00493699_m1&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minimal essential medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>11095-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trysin-EDTA solution(0.05%)</td>
			<td>Thermo Fisher</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome Spin Columns (MW3000)</td>
			<td>Thermo Fisher</td>
			<td align="right">4484449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td align="right">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applied Biosystems 7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transmission electron microscopes (TEM)</td>
			<td>JEOL Ltd</td>
			<td>120 kV TEM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus BX41 microscope</td>
			<td>Olympus</td>
			<td>BX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

激しい肺傷害 (アリ) と急性呼吸窮迫症候群 (ARDS)、高い罹患率と死亡率を持ち続けている肺疾患の異種グループを表します。アリの病態は優れているが定義されます。しかし、病気の複雑な性質のため分子療法まだ開発されなければなりません。ここで炎症反応のエクソソームの役割を記述するのに急性敗血症性肺傷害のリポ多糖 (LPS) 誘発マウス モデルを使用します。このモデルを使用して、マウス腹腔内組合にさらされているが miRNA とサイトカインは、炎症反応を調節するとパッケージ化された肺から気管支肺胞洗浄 (BAL) 液のエクソソームを分泌することを表示することができました。気管支上皮細胞タイト結合蛋白質の発現するエクソソーム マクロファージからリリースが中断を示す共培養モデル システムを使用してさらに、.炎症性応答に貢献する exosomal を往復させる生得免疫構造と細胞間クロストーク 1)、構造壁と 2) これらの Mirna ターゲットの混乱を治療する新しいプラットフォームを提供することが示唆します。ALI および ARDS。

敗血症性肺傷害を誘導して、マウスは、腹腔内の LPS (15 mg/kg) と扱われました。LPS 投与 24 時間以内は、図 1 aに示すように、好中球の流入が肺に見られました。エクソソームの隔離そして浄化に続いてマウス BAL 液が描かれました。BALF エクソソームの形態は、電子顕微鏡観察 (図 1 b) によって確認されました。
<p class="jov...

Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

リポポリサッカライド投与による敗血症性肺損傷における気管支肺胞洗浄エクソソーム

マウス腹腔内組合は、miRNAs とパッケージ化された気管支肺胞洗浄 (BAL) 液のエクソソームを分泌します。共培養システムを用いて、BAL 液中リリース エクソソームの気管支上皮細胞におけるタイト結合蛋白質の表現を中断させ、肺損傷を強調する炎症性サイトカインの発現を示す.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

bronchoalveolar-lavage-exosomes-lipopolysaccharide-induced-septic

Research

JoVE Journal

Genetics

10.8K ビュー.  Atlanta VAMC. マウス腹腔内組合は、miRNAs とパッケージ化された気管支肺胞洗浄 (BAL) 液のエクソソームを分泌します。共培養システムを用いて、BAL 液中リリース エクソソームの気管支上皮細胞におけるタイト結合蛋白質の表現を中断させ、肺損傷を強調する炎症性サイトカインの発現を示す.

ビデオ: Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

新たに単離したヒト絨毛細胞栄養芽層に対するsiRNAトランスフェクションおよびEMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

遺伝学

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

EDU染色によりショウジョウバエアダルト卵巣内のミトコンドリアDNA複製のその場の標識で

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

でヒストンminiSOGを使用した光遺伝学ランダム突然変異誘発<em&gt; C。エレガンス</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

プールされた shRNA MeCP2 再アクティブ化非アクティブ X 染色体上の画面

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

哺乳類細胞におけるメジャー クロマチン アクセシビリティ規制要素のホルムアルデヒドによる分離

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

クロマチンのループ構造の再編を CRISPR 媒介

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

南しみが付くことおよびポリメラーゼの連鎖反応を使用してマウス胚性幹細胞での相同組換えイベントの識別

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神経変性認知症予備モデルにおけるヒストン翻訳後修飾変化の特性

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

細胞遺伝学的手法を用いたタバココナジラミの卵の受精率を決定します。

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...