この作品は、NIH（EY012977）、バローズウェルカムファンド、ホワイトホール財団、スローン財団（AKM）から医歯薬学総合研究でのキャリア賞からの補助金によって可能となった。

1。ウイルスの取り扱いと保管<ol><li>適切な保護と取り扱い技術が使用されるウイルスの生物学的安全性レベルに基づいて選択する必要があります。これらの慣行は、CDCのウェブサイト上で利用可能な微生物学的および生物医学研究所第 5版、のバイオセーフティで見つけることができます。 （ <a href="http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm">http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm</a> ）。 AAVベクターの使用は、バイオセーフティレベル1（BSL - 1）が承認されています。ウイルスを処理する際にここに示されている実験では、白衣や手袋はBSL - 1のエージェントを処理するための手順に従って、着用されます。 </li><li>ウイルスの活動を維持するには、繰り返しの凍結融解を避けるために少量のアリコートに分割することをお勧めします。 </li><li>不要なオブジェクトのBSCをクリアし、70％エタノールで表面を殺菌することによってウイルスを小分けするためバイオセーフティキャビネット（BSC）クラスIIを準備します。 AAV -汚染された廃棄物を収集するためのフードに10％漂白剤溶液の入ったビーカーを置きます。また、BSCに入れ滅菌0.5mLを試験管とドライアイスの容器です。 </li><li> BSC外部の氷の上にウイルスストックを解凍。 </li><li>渦のウイルスとBSCの内部にチューブを開きます。 </li><li> 、0.5mLをチューブに必要な一定量の体積（例えば5μL）をピペットでチューブを閉じ、に急速冷凍ウイルスドライアイスに入れてください。 </li><li>すべてのウイルスが小分けされている場合、ウイルスのチューブと10％の漂白剤を含む廃棄物の容器にピペットチップを処分。 </li><li>フードから廃棄物のコンテナを削除し、さらに10％の漂白剤を追加してから、シンク下の漂白剤を注ぐ。あなたの研究所のバイオセーフティの役員の指示に従い、バイオハザード容器にプラスチック廃棄物を処分してください。 </li><li> 10％漂白剤で、ウイルスと接触したすべての機器または表面を清掃してください。手袋を捨てる。 </li><li> -80 ° Cの冷凍庫に保管してアリコート。 </li></ol> 2。手術<ol><li>吸収実験台の紙と外科領域をカバー。手術器具は、所属機関のバイオセーフティおよび動物使用のガイドラインに従い、無菌条件下で滅菌し、手術が行われる必要があります。 </li><li>ウイルスにマイクロピペットをロードするために専用される外科領域に隣接するエリアを選択し、吸収実験台の紙をかぶせます。このエリアの氷の容器にウイルスのアリコートを置き、そして手術が行われているとして、ウイルスは氷上で解凍することができます。 </li><li>ウイルスと接触すること等、ピペットチップの処分のために専用のウイルスの処理の領域で10％の漂白剤の廃棄物コンテナを設定します。 </li><li>約20ミクロンの先端の直径にガラスWiretrolのマイクロピペットを引き出します。マイクロピペットの平滑末端での鉱物​​油の小滴を置き、マイクロピペットで提供されるワイヤーのプランジャーを挿入します。 </li><li>マイクロポンプのアームのクランプでマイクロピペットを固定します。 </li><li>は0.5 mg / kgの用量におけるブプレノルフィン皮下マウスを注入する。 200 mg / kgの用量で腹腔内注射によりAVERTINとマウスを麻酔し、切開を入力してから毛を防ぐために十分な大きさの余白を残すことを確認しながら、外科はさみで頭の上から毛を取り除く。 </li><li>手術中C 37℃の体温を維持するためにstereotaxのベースに加熱毛布を添付しstereotaxにマウスを固定します。 </li><li>エリアを殺菌するためにエタノールとbetadineの三交互スクラブで頭を浸す。 </li><li>手術中に目の潤いを保つために各眼でTobradexの眼軟膏のドロップを置きます。 </li><li>頭の正中線の下切開を行い、頭蓋骨を露出させる背部皮膚を引っ張る。 </li><li>慎重に先の細いピンセットを用いて頭蓋骨から筋を取り除く。 </li><li>定位座標を用いて注入する領域を見つけ、外科ペンで頭蓋骨をマーク。 </li><li> 、頭蓋骨の亀裂まで1.4ミリメートルバリ、頭蓋骨の薄い領域に直径約2mmと歯科用ドリルを使用するいくつかのセグメントに薄くなった領域を分割する。 </li><li>この手順では、滅菌生理食塩水のアプリケーションと頭蓋骨の潤いを保つ。 </li><li>優しく極細先端鉗子を使用して間伐頭蓋骨セグメントを削除することにより、開頭術を行います。 </li></ol> 3。注射の準備<ol><li>蓋の上に配置キムワイプで、パラフィルムの小片の上にウイルスのウイルスとピペット1.5uL（1UL注射用）のチューブを開きます。 </li><li>ウイルスストックにマイクロピペットの先端を配置し、手動でプランジャーを撤回。難易度はマイクロピペットへのウイルスストックを描く経験している場合、先端はわずかにガラスの小さな部分を分割するマイクロピペットでキムワイプを貫通することが拡大することができます。 </li><li>ウイルスの少量まで、プランジャーの上に下マイクロポンプのアームは、マイクロピペットの先端から払拭される。綿の先端のアプリケーターを持つこの小さなドロップを削除し、WASTのアプリケーターを捨てるeのコンテナ。 </li><li>マイクロピペットは、脳に低くなるように目詰まりを防ぐために、マイクロピペットの先端に鉱物油の滴を適用します。 </li></ol> 4。ウイルスのインジェクション<ol><li> XとYの定位座標を用いて、注入される領域の上にマイクロピペットを置きます。この実験では、一次視覚野を特定するために使用される定位座標はブレグマに2.7ミリメートル後部と正中線に2.5ミリメートル外側です。非常にゆっくりと適切なZ位置に（1mm/1minuteのおおよその速度で）マイクロピペットを下げます。 </li><li> SYS Micro4マイクロポンプのコントローラのボックスに希望する注入パラメータを入力し、注入を開始する。この実験では、10分にわたって1マイクロリットルを注入の速度として使用されます。 </li><li>注入が終了すると、削除の際にウイルスの流出を防ぐために一から二分間休息するピペットを残す。この期間の後、非常にゆっくりと脳（上記と同じ率）からマイクロピペットを取り外します。 </li><li>頭皮を縫合し、組織の接着剤でそれを封印。は0.1 mg / kgの用量次の72時間にわたって毎に8〜12時間でブプレノルフィンの皮下動物を注入、または動物が痛みの兆候を展示している限り。それはambulatingとそのケージに戻される準備がされるまで、動物は熱ランプの下で回復することができます。イメージングの実験は、目的のインキュベーション時間（ウイルス注射後数日から数週間）の後に開始することができます。 </li></ol> 5。クリーン<ol><li> 10％漂白剤でマイクロピペットをすすぎ、鋭利物容器に捨てる。 </li><li>セクション1と同様に廃棄物容器に廃棄してください。 </li><li>バイオハザードビンに実験台の紙を捨て、10％の漂白剤とウイルスと接触している可能性がありますすべての表面や楽器を拭う。 </li><li>未使用のウイルスは、凍結/解凍サイクルは、ウイルスの劣化を引き起こすことを念頭に置いて、再び凍結されることがあります。 </li></ol> 6。代表的な結果<img src="/files/ftp_upload/2140/2140fig1.jpg" alt="図1" /> 図1は、。CMVプロモーターの制御下に緑色蛍光タンパク質（GFP）を運ぶ二本鎖アデノ随伴ウイルス血清型1（dsAAV S1）の注入後にニューロンを導入した。細胞体だけでなく、近位と遠位樹状突起は、落射蛍光顕微鏡を用いて画像の固定のセクションにはっきりと見える。スケールバー= 100μmの。 <img src="/files/ftp_upload/2140/2140fig2.jpg" alt="図2" /> 図2。ウイルスの拡散の程度だけでなく、標識された神経細胞、グリア細胞およびプロセスを示すdsAAV S1を使用して一次視覚野における頭蓋内ウイルスの注射の典型的な標識。スケールバー= 250μmの。 <img src="/files/ftp_upload/2140/2140fig3.jpg" alt="図3" /> 図3。dsAAVのS1を使用して海馬の細胞の標識。スケールバー= 250μmの。これらの数字はロウリーらを適合させたもの。2009 1

<ol>
	<li>Kaplitt, M. G., Leone, P., Samulski, R. J., Xiao, X., Pfaff, D. W., O'Malley, K. L., During, 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=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nat Genet. 8, 148-154 (1994).</li><li>Harding, T. C., Dickinson, P. J., Roberts, B. N., Yendluri, S., Gonzalez-Edick, M., Lecouteur, R. A., Jooss, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+gene+transfer+efficiency+in+the+murine+striatum+and+an+orthotopic+glioblastoma+tumor+model,+using+AAV-7-+and+AAV-8-pseudotyped+vectors.">Enhanced gene transfer efficiency in the murine striatum and an orthotopic glioblastoma tumor model, using AAV-7- and AAV-8-pseudotyped vectors.</a> Hum Gene Ther. 17, 807-820 (2006).</li><li>Lo, W. D., Qu, G., Sferra, T. J., Clark, R., Chen, R., Johnson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+transfer+to+the+brain:+duration+and+modulation+of+expression.">Adeno-associated virus-mediated gene transfer to the brain: duration and modulation of expression.</a> Hum Gene Ther. 10, 201-213 (1999).</li><li>Lowery, R. L., Zhang, Y., Kelly, E. A., Lamantia, C. E., Harvey, B. K., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+long-term+labeling+of+cells+in+the+developing+and+adult+rodent+visual+cortex+using+double-stranded+adeno-associated+viral+vectors.">Rapid long-term labeling of cells in the developing and adult rodent visual cortex using double-stranded adeno-associated viral vectors.</a> Dev Neurobiol. 69, 674-688 (2009).</li></ol>

動物と実験的なプロトコルは、実験動物の愛護ケアや使用に関するPHSポリシーに従い、動物資源（UCAR）上ロチェスター大学委員会の大学により承認された。

ウイルスを介した遺伝子デリバリーは、神経学的プロセスと脳疾患1,2,3の治療の研究のための大きな可能性を秘めている。この技術の偉大な多様性は、蛍光in vitroおよび in vivo 4 の両方のイメージングのための細胞を標識するために悪用される可能性があります。ここでは、神経細胞と強化された緑色蛍光タンパク質を発現する二本鎖アデノ関連ウイルスを用いた...

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<tr>
<td>Stoelting Mouse and Neonatal Rat Adaptor</td>
<td>&nbsp;</td>
<td>Stoelting</td>
<td>51625</td>
<td>Regular stereotax for securing animals for surgery may be substituted</td>
</tr>
<tr>
<td>Extra Fine Bonn Scissors, 8.5cm, straight tip, cutting edge 13mm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14084-08</td>
<td>&nbsp;</td>
<tr>
<td>Eye Dressing Forceps, 10cm, tip width 0.5mm, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11152-10</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5/45 Forceps- Dumoxel Standard Tip, 11cm, angled</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11251-35</td>
<td>Extra-fine tipped forceps for performing craniotomy</td>
</tr>
<tr>
<td>Standard Pattern Forceps, straight, 2.5mmx1.35mmtip, 12cm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11000-12</td>
<td>&nbsp;</td>
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<td>Microtorque Control Box and Tech2000 handpiece</td>
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<td>Ram Products, Inc.</td>
<td>TECH2000ON/OFF</td>
<td>Dental drill</td>
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<td>Micro Drill Stainless Steel Burrs 1.4mm tip diameter</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>19008-14</td>
<td>&nbsp;</td>
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<td>Wiretrol micropipettes, to deliver 1-5 Ul</td>
<td>&nbsp;</td>
<td>VWR International/ Drummond</td>
<td>5-000-1001 or 53480-287</td>
<td>&nbsp;</td>
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<td>Mineral oil</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>29447-338</td>
<td>&nbsp;</td>
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<td>Manual Micromanipulator and Tilting Base (right-handed)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>M3301-M3-R</td>
<td>Used for determining stereotaxic co-ordinates</td>
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<td>UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller</td>
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<td>World Precision Instruments, Inc.</td>
<td>UMP3-1</td>
<td>&nbsp;</td>
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<td>Sutures</td>
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<td>VWR International</td>
<td>95056-952</td>
<td>&nbsp;</td>
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<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Tobradex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Available from your institution&#x2019;s veterinary services</td>
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intracranial injection of adeno-associated viral vectors

蛍光タンパク質を発現するように設計ウイルスベクターの頭蓋内注射は、 インビボおよび脳切片の両方で異なる脳領域での細胞の特定のサブセットの可視化のための多目的な標識法です。 、蛍光色素の注入とは異なり、ウイルスのラベリングは、個々の細胞型を標的とトランスジェニックマウス系統を確立するよりも安価で時間がかかりますいます。この手法では、アデノ随伴ウイルス（AAV）ベクターは、定位座標、マイクロピペットと周囲の組織に与えるダメージを最小限にと希望する領域にAAVの正確な配達のための自動化されたポンプを用いて頭蓋内に注入されます。注入パラメータは、注射の注射、注入位置、注入量、速度、AAV血清型および遺伝子発現を駆動するプロモーターで動物の年齢を調整することによって、個々の実験に合わせて調整することができます。選択された条件に応じて、ウイルス誘発性遺伝子の発現がダウンして樹状突起棘のレベルまで、細胞を、個々の細胞や微細な細胞プロセスのグループの可視化を許可することができます。ここに示されている実験は、マウス一次視覚野の神経細胞とグリア細胞の標識のために緑色蛍光タンパク質を発現する二本鎖AAVの注入を示しています。

Watch this Scientific Journal Video about Intracranial Injection of Adeno-associated Viral Vectors at JoVE.com

Intracranial Injection of Adeno-associated Viral Vectors

ここでは、視覚野の神経細胞とグリア細胞の蛍光標識用AAVベクターの頭蓋内注射を提示する。

アデノ随伴ウイルスベクターの頭蓋内注射

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

intracranial-injection-of-adeno-associated-viral-vectors

Research

JoVE Journal

Neuroscience

47.1K ビュー.  University of Rochester. ここでは、視覚野の神経細胞とグリア細胞の蛍光標識用AAVベクターの頭蓋内注射を提示する。

ビデオ: Intracranial Injection of Adeno-associated Viral Vectors

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

薬の配達のためのMicroinjections、ウイルスベクター、または細胞移植で脳深部構造のターゲティング

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

神経科学

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

マウス網膜色素上皮における遺伝子治療用ウイルスベクターの縁アプローチ、網膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

成体マウスにおける子宮頸部アデノ随伴ウイルス9（AAV9）ベクター送達

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

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 ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

成体マウスの遺伝子組換えアデノ随伴ウイルスの直接髄腔内注入

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

生産・精製・ アデノ随伴ウイルスを用いたベクトルの品質管理

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

オンラインMRI画像のガイダンスの下でレソスマカクの皮質への光遺伝学アデノ関連ウイルスベクターの対流強化された配信

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

ウイルスベクターの脳内注射によるインビボグリア細胞のインターニューロンへの直接リプログラミング

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

マウスにおけるコフィリンを標的とするアデノ関連ウイルスを用いて操作した大脳皮質領域の電気コルチコチコ

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.