NIH R01MH084894は、この研究の資金調達に参加しました。我々は、博士に感謝したいと思います。マウスの寄付と、この作品の撮影時に、その手術や行動施設の利用のためのマウントサイナイ医科大学でヤスミンHurd氏とスコット・ルッソ。

注：動物の飼育や心配事のためのすべての手順は、マウントサイナイ医科大学の施設内動物管理使用委員会（IACUC）規則に従って行いました。作業中は滅菌手袋を使用してください。 1.薬剤およびウイルスの準備<ol><li> 医薬品の準備 <ol><li> 0.9％の食塩水の12.9ミリリットルで100 mg / mlでケタミンおよび20mg / mlのキシラジンの0.75ミリリットルの1.35ミリリットルを溶解することにより15.0ミリリットルのケタミン/キシラジン麻酔薬を準備します。溶液を十分に混合します。 </li></ol></li><li> ウイルス調製物 <ol><li> mGlu2のクローンを作成し、mGlu2ΔTM4Nは、標準的なプロトコルは、以前に6を説明し、次のシストロン性単純ヘルペスウイルス（HSV）ベクター内に構築します。以前6,13,14に記載されているようにウイルス粒子をパッケージ化します。残基の置換アラ-677 4.40、mGlu2でのAla-681 4.44およびAla685 4.48 Ser686 4.40、pHについてE690 4.44とのGly-694 4.48 mGlu3（HA-mGlu2ΔTM4N）に以前6に記載されています。 注：これは、以前にキメラ構築HA-mGlu2ΔTM4Nが無傷のGタンパク質依存6シグナル伝達と細胞膜で発現することが示されました。 </li><li> C使用しないときには-80℃でウイルスベクターを保管してください。氷の上でウイルスベクターを解凍してから、10μlのアリコートに分注し。外科的手順については、氷上に保ちます。 </li></ol></li></ol> 2.手術<ol><li> 手術の準備 <ol><li>マウスを計量し、（詳細については、1.1.1を参照）をケタミン/キシラジンのカクテルの適切な用量でマウスを注入。 </li><li>適切に麻酔をかけた場合、疼痛応答のための足と尾を絞る参照するには、マウスをチェックし、応答を誘発することができない場合は、マウスが適切に麻酔されます。 </li><li>バリカンを使用して、鼻の先端に頭蓋底からマウスの頭を剃ります。マウスAFに眼科ゲルを適用しますマウスの失明を防ぐためにterward。 </li><li>定位フレーム上にそれぞれ注射器をロードします。彼らは10度法線から離れているように、そして、定位フレームの各アームの垂直部分を傾けます。針がお互いに直面しているように、アームが傾いていることを確認してください。 </li><li> 70％エタノールで針を充填することにより、各注射器を清掃してください。注射器がクリーンであることを確実にするために、針に少なくとも3回を埋めます。 </li><li>針が洗浄された後、二重蒸留H 2 Oで針を充填することによって針をフラッシュフラッシュたら、二重蒸留H 2 Oの1.3μlの各針を埋めます二重蒸留H 2 Oの0.3μLを解放するために、シリンジのプランジャーをねじります針の先端での水のビーズは、慎重に水を拭き取る場合。何も注射器から出てきていない場合は、完全にダウンしてプランジャーを押し、その後、注射器の洗浄を繰り返します。 </li><li>水を充填した後、次いで、シリンジを充填注射器を引き上げ空気の0.5μlの。 </li><li>空気や水が注射器に入ると、慎重にウイルス液の1.3μlのシリンジを埋めます。この時点で、シリンジ内の総容量は2.8μlであることを確認してください。再びウイルスの0.3μLを解放するために、注射器の先端をねじります。針の先端の液体ビーズは、慎重に液体を拭き取る場合。何も注射器から出てきていない場合は、完全にダウンしてプランジャーを押し、その後、注射器の洗浄を繰り返します。 </li></ol></li><li> 手術 <ol><li>頭蓋骨が水平かつ平坦になるように、定位フレームを調整することを確認して、定位フレームにマウスを接続します。露出した頭皮にpovodine - ヨウ素を適用します。メスを用いて、露出した剃ったエリア内の頭蓋骨の正中線に沿って矢状切開を行います。その後、頭蓋骨が露出したままであることを確認するために切開部位の皮膚にビュレットクランプを取り付けます。 </li><li>の縫合糸を露出させるために骨膜を溶解除去するH 2 O 2を使用して頭蓋骨。今ブレグマおよび縫合糸が見えること、頭蓋骨が水平であることを確認するために、定位フレームを調整するようにしてください。 </li><li>ブレグマと注射器の針の先端の位置を合わせ、ブレグマの座標を記録します。針が挿入されようとしているの座標を計算します。 <ol><li>吻側-Cauldal（RC）面については、記録されたRCのブレグマ座標（ブレグマから1.6）に1.6ミリメートルを追加します。背腹（DV）面については、記録されたDVのブレグマ座標（ブレグマから-2.4）から2.4ミリメートルを引きます。 </li><li>最後に、内側側横方向（ML）面のために、記録されたMLのブレグマ座標（ブレグマから2.6）に2.6ミリメートルを追加します。これは二国間の注射であるように、すべての座標は、左右両方の座標を記録するようにしてください。 </li></ol></li><li>希望の座標に針を持参してください。針を挿入し、ドリルで、マークされた領域をドリルダウンしようとしている場所の場所をマークします。 </li><li>綿の先端アプリケータのwiと余分な血液や骨片を離れPE。 </li><li>針の先端は、脳の表面に触れている頭蓋骨に針を持参してください。その後、ゆっくりとそれらを下げる目的の座標に針を下げます。 </li><li>針は、所望の座標になったら、ゆっくりと（合計0.5μLで）5分にわたって毎分針0.1μlとプランジャーをねじることによって、注射器の内容物を注入します。 </li><li>注入が行われた後、別の5分間の皮質に注射器を残します。 </li></ol></li><li> アップクローズ/ケア <ol><li>着実に、ゆっくりとマウスの皮質から針を外します。そして、定位フレームからマウスを削除します。 </li><li>切開部から皮膚のベースフラップにシアノアクリレート（皮膚接着剤）を塗布した後、ピンセットで皮膚のフラップをつかみ、それらを一緒に配置します。 </li><li>シアノアクリレートが乾燥することができます。加熱パッドの上にケージにマウスを置きます（加熱パッドはオプションです外科場合それ以外の場合は不要） - iCalのスイートは、37℃の室温下に保たれています。寝具は、手術部位に付着しないことを確認するために、ペーパータオルの上にマウスを置くようにしてください。 </li><li>処置後60分 - 外科処置の長さに応じて、マウスが30以内に麻酔から外れていることを確認してください。手術後、動物はそれ自身のケージに入れ、それを回復するためにその部屋に戻る前に、意識的になるまで監視されています。彼らは脳の生化学的経路の一部を変更することによって、私たちの実験の結果を変更することができるので、無鎮痛薬は、手術後に使用されていません。しかし、彼らは手術の日に、麻酔から回復するまで、動物が監視され、感染および痛み/苦痛の評価の徴候について毎日術後。 </li></ol></li></ol> 3.ヘッド単収縮応答実験<ol><li> セットアップ <ol><li> 3日stereota後 - 2、午前10:00と午後2:00の間のすべての行動試験を実施ウイルス粒子のCTIC注射。 </li><li>ディゾルブ（±）-1-（2,5-ジメトキシ-4-ヨードフェニル）2.0ミリグラム/ kgの0.9％生理食塩水に-2-アミノプロパン塩酸塩（DOI）。また、0.9％生理食塩水溶液を調製。 </li><li>任意の寝具なしホームケージ（28×18×15センチ）を準備し、三脚を使用して、ビデオカメラのビューが直接ホームケージの上にあるようにカメラを調整します。 </li><li>実験の開始に先立って、少なくとも4時間、部屋にマウスを慣らします。 </li><li>ヘッド単収縮を記録するビデオカメラを設定します。 </li></ol></li><li> 実験 <ol><li>それはホームケージの真上になるようにカメラを置きます。全体のホームケージが視野に入るようにカメラを調整します。 </li><li>マウスを計量し、0.9％生理食塩水またはDOI（0.01ミリリットル/ g）のいずれかの適切な用量で腹腔内にマウスを注入します。注：マウスは25グラムの重量を量る場合、0.25ミリリットルの全容積に用量を投与。 </li><li> foのホームケージに戻って各マウスを置きますR 10分間。 10分後、空のホームケージの中央にマウスを置いて、カメラの視野内に死角がないことを確認します。ビデオカメラを押してレコード。部屋のままにしておきます。 注：マウスの動きやその中の様々な行動反応（。。。頭がけいれん、耳の傷などは DOIによって誘発される行動応答の完全なリストについてのゴンザレス-Maeso ら 2007補足データテーブル1を参照してください）3、のために記録されます30分。したがって、記録された視野内に死角が存在しないことが重要です。 </li><li> 30分には元のホームケージにバックカメラと場所マウスの録音を停止した後。各マウスについて、このプロセスを繰り返します。 </li></ol></li><li> レビュー <ol><li>テープは、マウスの実験条件に盲目各審判のレビューを持っている）（ すなわち 、ヘッドの間に使用される薬剤は、実験や頭蓋内注射時に使用されるウイルスをけいれん）。手動ですべてのヘッドがthrougけいれん記録ビデオハウト。 注：ヘッド単収縮は、マウス（補足ビデオ）が実施し、迅速な振とうヘッドの動きとして定義されています。 </li><li>各マウスについて、ブラインド審判の合計3からの最終的なHTR応答を平均します。実験条件によってその後のグループ、これらの値を、統計分析を行う（ すなわち 、t検定またはANOVA）。 </li></ol></li></ol>

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O., Persani, L., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+by+internalized+G-protein-coupled+receptors.">Signaling by internalized G-protein-coupled receptors.</a> Trends Pharmacol Sci. 31 (5), 221-228 (2010).</li><li>Celada, P., Puig, M. V., Diaz-Mataix, L., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallucinogen+DOI+reduces+low-frequency+oscillations+in+rat+prefrontal+cortex:+reversal+by+antipsychotic+drugs.">The hallucinogen DOI reduces low-frequency oscillations in rat prefrontal cortex: reversal by antipsychotic drugs.</a> Biol Psychiatry. 64 (5), 392-400 (2008).</li><li>B&#233;&#239;que, J. -. C., Imad, M., Mladenovic, L., Gingrich, J. A., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+5-hydroxytryptamine+2A+receptor-mediated+facilitation+of+synaptic+activity+in+prefrontal+cortex.">Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (23), 9870-9875 (2007).</li></ol>

The authors have nothing to disclose.

一緒にmGlu2-KOマウス5の前の所見と、培養細胞において5-HT 2A -mGlu2受容体複合体を形成しないmGlu2およびmGlu2 / mGlu3キメラ構築物を用いた結果は、5-HT 2A -mGlu2ヘテロマー受容体複合体ことを示唆していますマウスの前頭皮質は、LSDのような幻覚5-HT 2A受容体アゴニストによって頭部攣縮挙動を誘発するために必要とされます。この方法の制限は、それが天然の組織内...

そのようなLSD、サイロシビンやメスカリンなどの幻覚剤は、人間の意識、認知と感情7-9に大きな変化を引き起こします。セロトニンの不活性化は、遺伝的または薬理学的のいずれかの方法により、5-HT 2A受容体のシグナル伝達は、両方の齧歯類モデル3,10、およびヒト11で幻覚剤に著しく減衰行動反応を引き起こします。幻覚剤は、他の受容体サブタイプ8に結合するが、5-HT 2A受容体は、これらの化学物質の固有の行動活性に必要と考えられています。 グループII代謝型グルタミン酸受容体（ すなわち 、mGlu2およびmGlu3）は幻覚剤の分子機構に関するかなりの注目の対象と精神病12の基礎となる彼らの重要な役割となっています。これまでは、mGlu2タンパク質の発現なしでのマウス（mGlu2-KOマウス）はHALの細胞や行動への影響に鈍感であることが実証されていますlucinogens 5。また、5-HT 2AおよびmGlu2受容体は、セロトニンおよびグルタミン酸リガンドが生細胞1,2のGタンパク質結合のパターンを調節し、それを通して、特定のヘテロマー複合体を形成することが示唆されています。 構造的には、膜貫通（TM）ドメイン4およびmGlu2の5は、5-HT 2A受容体5とヘテロマー形成に重要な役割を果たしています。さらに、さらなる調査がmGlu2のTM4の細胞内の端部に位置する3つの残基が、細胞を6生体内5-HT 2A -mGlu2レセプターヘテロを形成するのに必要であることを実証しました。 異種発現系で観察されたこれらの知見に基づいて、ここでは、5-HT 2Aとの間のヘテロマー形成するかどうかをテストするために、野生型mGlu2およびmGlu2-KOマウスの前頭皮質におけるmGlu2 / mGlu3キメラ構築物のHSV媒介表現の使用を記載していますそして、mGlu2はのために必要です幻覚5-HT 2A受容体アゴニストによって誘導される頭部攣縮挙動。

<table><tbody><tr><td>mGlu2 bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>mGlu2&amp;Delta;TM4N bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>GFP bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Lloyd</td><td>List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Vedco</td><td>KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ophthalmic gel</td><td>Fisher Scientific</td><td>NC0550805</td><td></td></tr><tr><td>Burret clips</td><td>Fisher Scientific</td><td>NC9268369</td><td></td></tr><tr><td>Feather surgical blade</td><td>Fisher Scientific</td><td>NC9032736</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Scientific</td><td>19-898-919&amp;nbsp;</td><td></td></tr><tr><td>Hamilton syringe</td><td>Fisher Scientific</td><td>14815203</td><td></td></tr><tr><td>Hamilton&amp;trade; Small Hub Removable Needles (33 Ga)</td><td>Fisher Scientific</td><td>14816206</td><td></td></tr><tr><td>Cordless Micro Drill</td><td>Fisher Scientific</td><td>NC9089241</td><td></td></tr><tr><td>Dermabond Dermal Adhesive</td><td>Fisher Scientific</td><td>NC0690470</td><td></td></tr><tr><td>(&amp;plusmn;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td><td>Sigma-Aldrich</td><td>42203-78-1</td><td>Dissolved in 0.9% saline solution to the concentration of 2.0 mg/kg</td></tr></tbody></table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

以前の知見は、ヘッド単収縮マウスの行動応答を確実かつロバスト幻覚剤によって誘発され、それは、5-HT 2A -KOマウス3には存在しないことを示しています。また、DOIとLSD幻覚の5-HT 2Aアゴニストによって誘発される頭部攣縮応答が有意mGlu2-KOマウス5に減少したことが示されました。しかし、以前の知見は、説得力の5-HT 2Aおよびm...

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

この記事では、GPCRヘテロマーの形成を必要とする行動アッセイをテストするために、マウスの前頭皮質にウイルスベクターを注入する方法について説明します。

マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Research

JoVE Journal

Neuroscience

7.3K ビュー.  Icahn School of Medicine at Mount Sinai. この記事では、GPCRヘテロマーの形成を必要とする行動アッセイをテストするために、マウスの前頭皮質にウイルスベクターを注入する方法について説明します。 

ビデオ: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9 仲介の対象となる統合生体内で相同性を介した終わり参加ベースの戦略を使用して

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

遺伝学

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

レンチ ウイルス媒介トランスジェニック マウスの生産: シンプルで高効率な創設者の直接の研究法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅神経細胞のレンチウイルスを介した遺伝子操作と可視化 in vivoで</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

神経科学

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

マウスにおける再発性ヘルペス性間質性角膜炎、人間のHSKを研究するためのモデル

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

免疫・感染学

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

遺伝的に定義された神経回路のトレースウイルス

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

マウス脊髄における条件遺伝子操作のためのウイルスベクターの定位注射

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest.
The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

in vivoマイクロインジェクションとエレクトロポレーションを用いた体細胞ゲノム改変マウスモデル

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. ...

がん研究

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

マウス角膜と結膜のライブイメージングのためのカスタム多光子顕微鏡プラットフォーム

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

生物工学

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

アデノ随伴ウイルスを介する遺伝子発現により脊髄のニューロンを遺伝的に定義されています。

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

<meta charset="utf8"></head><body>Th17分化における遺伝子編集のためのガイドRNAのレトロウイルス形質導入

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

<meta charset="utf8"></head><body>in vitroでのTh17分化の研究のためのレトロウイルスCRISPR/Cas9媒介遺伝子ターゲティング

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

in vitroでのTh17分化の研究のためのレトロウイルスCRISPR/Cas9媒介遺伝子ターゲティング

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現

要約

さらに動画を探す

CRISPR/Cas9 仲介の対象となる統合生体内で相同性を介した終わり参加ベースの戦略を使用して

レンチウイルス媒介トランスジェニックマウスの生産: シンプルで高効率な創設者の直接の研究法

嗅神経細胞のレンチウイルスを介した遺伝子操作と可視化 in vivoで

マウスにおける再発性ヘルペス性間質性角膜炎、人間のHSKを研究するためのモデル

遺伝的に定義された神経回路のトレースウイルス

マウス脊髄における条件遺伝子操作のためのウイルスベクターの定位注射

in vivoマイクロインジェクションとエレクトロポレーションを用いた体細胞ゲノム改変マウスモデル

マウス角膜と結膜のライブイメージングのためのカスタム多光子顕微鏡プラットフォーム

アデノ随伴ウイルスを介する遺伝子発現により脊髄のニューロンを遺伝的に定義されています。

in vitroでのTh17分化の研究のためのレトロウイルスCRISPR/Cas9媒介遺伝子ターゲティング

マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現

要約

さらに動画を探す

CRISPR/Cas9 仲介の対象となる統合生体内で相同性を介した終わり参加ベースの戦略を使用して

レンチ ウイルス媒介トランスジェニック マウスの生産: シンプルで高効率な創設者の直接の研究法

嗅神経細胞のレンチウイルスを介した遺伝子操作と可視化 in vivoで

マウスにおける再発性ヘルペス性間質性角膜炎、人間のHSKを研究するためのモデル

遺伝的に定義された神経回路のトレースウイルス

マウス脊髄における条件遺伝子操作のためのウイルスベクターの定位注射

in vivoマイクロインジェクションとエレクトロポレーションを用いた体細胞ゲノム改変マウスモデル

マウス角膜と結膜のライブイメージングのためのカスタム多光子顕微鏡プラットフォーム

アデノ随伴ウイルスを介する遺伝子発現により脊髄のニューロンを遺伝的に定義されています。

in vitroでのTh17分化の研究のためのレトロウイルスCRISPR/Cas9媒介遺伝子ターゲティング

レンチウイルス媒介トランスジェニックマウスの生産: シンプルで高効率な創設者の直接の研究法