この研究は、ソウル大学研究助成（800から20140542）によってサポートされていました、NRF / MESTのパイオニア研究プログラム（2012から0009544）、およびNRF / MESTの生体信号解析技術イノベーションプログラム（2009から0090895）、とNRF / MESTのグラント（2015M3A9E6028949）。

動物の全ての実験は、眼科と視覚研究における動物の使用のためのビジョンと眼科文の研究のための協会に従って実行、およびガイドラインや規制は、ソウル国立大学施設内動物管理使用委員会とソウルで定められました大学病院バイオセーフティ委員会。 1.インジェクションキットおよびウイルスベクターを準備<ol><li>酸化エチレンガスを使用して滅菌33 G鈍針を備えたマイクロシリンジを準備します。マイクロチューブ（ すなわち、1×10 6 TU /μL）で十分な力価のために、PBS中のウイルスベクターを希釈します。シリンジ内の任意のデッドスペースを除去するために、ウイルスベクターを用いて、シリンジを数回フラッシュします。 </li></ol>ウイルスベクターの2網膜下注射<ol><li> tiletamiの混合物の腹腔内注射で-成体マウス（8週齢、すなわち、6）麻酔NE及びゾラゼパム（1：1、2.25 mg / kg体重）及びキシラジン塩酸塩（0.7 mg / kg体重）、または代替の適切な麻酔レジーム。 </li><li>フェニレフリン0.5％の点眼で瞳孔を拡張し、0.5％のトロピカミド。 </li><li>ウイルスベクター2μlの - 1.5でロードすることにより、マイクロシリンジを準備します。 </li><li>まぶたを開いて、便利な注射のための赤道を露出させ、手術用顕微鏡下に集中するために目を突出しています。注入を終えまで突出目の位置を維持する、または針の変位が注入中に発生する可能性があります。しっかりと眼球を保持するために、眼窩縁の外側に指を置きます。 </li><li>角膜表面への眼科用粘弾性溶液の液滴を適用します。 </li><li>網膜を視覚化するために、角膜の上に小さな丸いカバースライドを置きます。 </li><li>さらに網膜下注射のための無菌の30 G 1/2針を使用して角膜縁にわずかに後方に小さな穴を穿刺。劣っFO穴を作りますR右目、利便性のために、左眼のための優れました。 注：穴が時間的または経鼻で行われる場合、それは注射後まぶたによって覆われるのは難しいです。初期パンクはわずか輪部に沿って実行して、容易に認識することができ輪部血管を回避するために、角膜輪部に後方なされるべきです。最初の穿刺をしながら針でレンズを打つないように注意してください。レンズ穿刺を回避するために、針の全体の傘を挿入しないでください。 </li><li>プレパンクチャーホールを介してマイクロリットルの注射器の33 G鈍針を置き、穏やかな抵抗が感じられる点まで、網膜下腔に針を近づけます。 注：網膜下注射のために、針の最良のアプローチ角は、虹彩面に対して約45度であり、鈍い針は乳頭周囲の領域に向かって後方に押されるべきです。点線の四角は網膜下の充血のために硝子体腔を横切る示唆針経路を示しました図1中のn。 注：彼らは非常に柔らかいようピアス（または通過）網膜層ときに抵抗が感じないがあるでしょう。このように、軽度の抵抗の最初の気持ちは、針が既にRPE層に触れた、針の挿入が停止されるべきであることを示しています。針は、潜在的な網膜下腔に配置する必要がありますので、過度の圧力と強膜組織を貫通しないように注意してください。針が過大な力で強膜組織を穿刺した場合、それは抵抗なく外眼軌道の空間に入ります。 （このステップは非常に重要です） </li></ol><img alt="図1" src="/files/ftp_upload/53030/53030fig1.jpg" /> 網膜下注射の図1の模式図で。H＆Eは、マークされた網膜下注射用の針経路（点線の四角）との構造を描いたマウスの眼の断面を染色しました。倍率：40X、スケールバー：<html> 500ミクロン。 <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a> <ol start="9"><li>不要な組織の損傷を避けるため、ゆっくりと針を撤回する震えずに網膜下の空間に静かにウイルスベクター（ すなわち、1×10 6 TU /μl）を注入します。 2.4で説明したように、注射の際にしっかりと眼球を保持します。 </li><li>何の網膜出血がないことを確認する手術用顕微鏡下で注入後の網膜下ブレブの形成を観察します。 </li></ol><img alt="図2" src="/files/ftp_upload/53030/53030fig2.jpg" /> 図2.網膜出血せずに網膜下Belb形成。手術用顕微鏡下での網膜下注射後の網膜下belbの顕微鏡図は、網膜出血のない成功ブレブ形成を示します。M /ファイル/ ftp_upload / 53030 / 53030fig2large.jpg「ターゲット= &quot;_空白&quot;&gt;この図の拡大版を表示するには、こちらをクリックしてください。 <ol start="11"><li>静かに自己封止用注射部位をカバーするために瞼を閉じます。ホームケージにマウスを返し、評価まで生き続けます。 注：研究者は手順に慣れた後、彼らはこれらのステップをスキップすることにより、迅速な手順で再現性のある結果を得ることができます（2.5、6、および10） </li></ol>網膜色素上皮における遺伝子送達の有効性の評価3。 <ol start="1"><li> CO 2チャンバー内でマウスを生け贄に捧げます。はさみを使用して、目を摘出。 </li><li>数日に最低1時間、4℃で4％パラホルムアルデヒド溶液中で目を修正しました。 </li><li>細かい鉗子で角膜をつかみ、マイクロはさみで角膜を穿刺。角膜輪部に沿ってシザリング後の角膜を削除します。ピンセットでレンズを取り外してください。 </li><li> mの毛様体トリムicro-はさみ網膜剥離を可能にします。静かRPE /脈絡膜/強膜複合体からの全網膜を引っ張ります。フラットマウント用4〜8葉を作るために視神経に近い中心に周囲から放射状に数回RPE /脈絡膜/強膜複合体をカットします。 </li><li>ダウンRPE側を行い、筋肉、結膜および視神経を含む強膜側の残りのすべての組織をトリミングするRPE /脈絡膜/強膜複合体を反転します。残りの組織は複雑な凹凸を作るため、この手順は、フラットRPE /脈絡膜/強膜複合体を作ることが重要です。 </li><li> （詳細な方法のための参考文献を参照して、オプション）特定の研究目的のために一次および二次抗体との複合体をインキュベートします。スライドガラス上のRPE /脈絡膜/強膜複合体をフラットマウントし、吸収性スポンジを含むPBSを吸収します。 </li><li>取付溶液30μlを添加して、カバースライドを配置します。フルオレセイン顕微鏡下でベクター送達の有効性のためのサンプルを観察します。でサンプルを保ちますさらに観察用冷蔵庫。 </li></ol>

<ol>
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The authors have nothing to disclose.

このビデオの記事では、RPE /脈絡膜/強膜フラットマウントの代表的な結果で詳細に輪部-アプローチ網膜下注入技術を説明しました。これは、RPEへのウイルスベクターの網膜下注射のための簡単​​で便利な技術です。注入時のブレブ形成の直接可視化は初心者のための正確な配達のための重要なステップです。可視化実験8-10誌で紹介し、いくつかの網膜下注入技術があります。網膜...

眼科では、遺伝子治療は、単一遺伝子継承網膜症における治療法として浮上しています。レーバー先天性amurosis 1,2、網膜色素変性症3、および先天性脈絡膜欠如4を含む網膜色素上皮（RPE）における遺伝子に関連した継承された網膜症があります。遺伝子治療の研究分野は、前臨床研究や、 アデノ -関連ウイルス（AAV）、 レンチウイルス （LV）およびアデノウイルス （AD）などのウイルスベクターを用いた臨床試験の両方に拡大している5。別のウイルスベクターは、網膜内の異なる親和性を有しています。安全かつ効果的な遺伝子治療のために、ウイルスベクターは、注意深く標的細胞および標的遺伝子に応じて選択されるべきです。 遺伝子送達の経路は、従って、それは慎重に同様に選択されるべきで、また、細胞を標的とする効果的な遺伝子送達のために重要です。ウイルスベクターの眼内送達のための2つの最も一般的な方法は、subretされていますinal注射や硝子体内注射6。後者は、硝子体内注射は、広く滲出型加齢黄斑変性症（AMD）および糖尿病性網膜症7の黄斑浮腫で脈絡膜血管新生を治療するための薬物送達のために使用されています。硝子体内経路は、硝子体と内側の網膜へのウイルスベクターの露出を提供していますが、外側の網膜へのベクターの拡散が制限されています。一方、網膜下の経路は、ローカライズ胞を誘導する、網膜とRPEの間の潜在的な空間へのウイルスベクターの直接送達を提供します。したがって、網膜下注射は、現在、光受容体細胞およびRPEを標的とする、より効率的なルートであると考えられます。外科的アプローチの面では、プラナは、ヒト患者における網膜の損傷を避けるために硝子体内注射のための安全なエリアとして選択されているPARS。単にマウスにこの方法を変更することによって、私たちは輪部アプローチを介して、網膜下またはintravireallyウイルスベクターを注入することができます。 このビデオで記事は、我々は、マウスのRPEへのウイルスベクターの網膜下注射の簡単で便利な方法を示しています。 30 G 1/2針で角膜輪​​部の後方に単一の穿刺後、33 G鈍針搭載マイクロリットルのシリンジは輪部穿刺部位を介して網膜下腔に挿入されます。 1.5のウイルスベクター - 2μlのボリュームが網膜下ブレブを誘発網膜とRPEとの間の潜在的な空間に注入されます。この手順では、手術用顕微鏡を用いて直接可視化の下で行うことができます。反復練習も、ブレブ形成の直接可視化することなく、複製の結果を保証します。これは、研究者がマウスのRPEにおける網膜下の遺伝子送達のために、正確かつ時間節約実験を行うのに役立ちます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TWEEZERS DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm TIPS</td>
			<td>WPI</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>VANNAS Scissors S/S, 105mm</td>
			<td>WPI</td>
			<td>555583S</td>
			<td></td>
		</tr>
		<tr>
			<td>33G Blunt needle</td>
			<td>WPI</td>
			<td>NF33BL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil Syringe, 10 microliter&#160;</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE-KIT</td>
			<td>WPI</td>
			<td>RPE-KIT</td>
			<td>For easy one hand injection</td>
		</tr>
		<tr>
			<td>30Gx1/2 (0.3mmx 13mm) BD PrecisionGlideTM Needle</td>
			<td>BD</td>
			<td>305107</td>
			<td>Initial puncture for subretinal injection</td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses (No. 1 3 mm diameter)</td>
			<td>Warner Instruments</td>
			<td>64-0720&#160; (CS-3R)</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica operating microscope</td>
			<td>Leica</td>
			<td>LM M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresecein microscope</td>
			<td>Nikon</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>Thermo scientific</td>
			<td>TMO.LV-Ctr</td>
			<td>Used to dilute vectors</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td>Used to dilute vectors</td>
		</tr>
		<tr>
			<td>Troperin (Phenylephrin 0.5%-Tropicamide 0.5%)</td>
			<td>Hanmi</td>
			<td></td>
			<td>For dilation</td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile)</td>
			<td>Bausch&#38;Lomb</td>
			<td></td>
			<td>For topical anesthesia</td>
		</tr>
		<tr>
			<td>Healon GV OVD</td>
			<td>Abbott Medical Optics Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride)</td>
			<td>Virbac</td>
			<td></td>
			<td>For general anesthesia</td>
		</tr>
		<tr>
			<td>Rompun&#174; injection (Xylazine HCl)</td>
			<td>Bayer</td>
			<td></td>
			<td>For general anesthesia</td>
		</tr>
	</tbody>
</table>

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.

このプロトコルにより、ウイルスの遺伝子導入の網膜下注射の有効性を評価するために、我々は、インジケータ用GFPおよびRFPの両方を発現するCMVプロモーターを有する市販のLVベクターを使用していました。目は研究目的に応じて適切な期間の後に摘出しました。代表的な結果を得るためには、目が網膜下注射後10週および20週に摘出しました。上述の方法を用いて網膜を完全に除去した後、R...

Watch this Scientific Journal Video about Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium at JoVE.com

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

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 ...

limbal-approach-subretinal-injection-viral-vectors-for-gene-therapy

Research

JoVE Journal

Neuroscience

20.9K ビュー.  Seoul National University College of Medicine. 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.

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

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

網膜色素上皮生理学と病態の研究のための実験的モデル

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

神経科学

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

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 ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

網膜色素上皮の全マウントするためのマウスの眼の解剖

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

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 ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

永続かつ広範な神経伝達のための新生児マウス脳の脳室内ウイルス注射

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

マウスにおける経頭蓋 Photobiomodulation 療法のプロトコル

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

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 ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

無条件網膜色素上皮を用いた、オルガマウス網膜の長期無血清栽培

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Viral Construct (AAV) Injection in Mouse Superior Colliculus for Calcium Imaging

Begin by securing the anesthetized mouse onto a stereotaxic frame with ear bars. Apply ophthalmic ointment on the mouse's eyes to prevent drying, and maintain the body temperature of the mouse at 37 degrees Celsius with a thermostatic heating pad. After shaving the fur and sterilizing the surgical site, inject lidocaine, tilidine, and meloxicam.Once the skin over the SC is removed, clean the skull with a cotton swab. Adjust the stereotaxic frame until the skull surface is flat. Using a drill, create a whole 0.5 millimeter lateral, and 0.42 millimeter anterior to the lambda, until the dura is exposed.Inject an adeno-associated virus, or AAV, expressing GCaMP6m, at depths of one millimeter and 1.6 millimeters from the Lambda using a beveled glass micro-pipette. After the injection, slowly withdraw the injector and proceed with the implantation of the head plate.

カルシウムイメージングのためのマウス上結腸へのウイルスコンストラクト(AAV)注射

CNS遺伝子治療のための幼若ラットにおけるAAV9の腰椎穿刺送達

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

著者スポットライト:若年ラットにおける髄腔内遺伝子治療の有効性の評価

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

腰椎貯水槽注射による幼若ラットの髄腔内ベクター送達

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...