We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

在这项研究中使用的所有动物根据ARVO声明动物在眼科和视觉研究中使用的照顾和处理。 1.生产重组腺相关病毒（腺相关病毒）由HEK-293细胞17,18的瞬时转染的注:请参阅麦克卢尔C，朱庇特（2011）19。 <ol><li>纯化大规模质粒制备的AAV载体质粒（至少1毫克/毫升）。使用3质粒。所述AAV辅助质粒携带复制和衣壳基因 ​​未经AAV反向末端重复的ITR，所述转基因表达盒携带AAV ITR称为转移质粒，并且该质粒供给腺病毒辅助基因的E2，E4和VA RNA基因所指为pHELPER。 </li><li>转染293细胞使用聚乙烯亚胺（或其他转染试剂）这3个质粒。高于80％的转染效率是必要的良好的六RAL产量。 </li><li>净化从293细胞裂解液重组AAV上的碘克沙醇梯度。 15％，25％，40％，和60％的碘克沙醇被用于获得梯度。收集含AAV颗粒的40％的碘克沙醇馏分，浓缩超速离心后的级分在500000×g离心对PBS与0.001％的Pluronic 1小时，缓冲液交换（磷酸盐缓冲盐水）。 </li><li>要确定病毒基因组滴度20，进行DNA酶蛋白酶K消化后QPCR。正向和反向引物扩增位于AAV2 ITR的62 bp的区域。对于质粒的标准，该探针用FAM和具有黑孔猝灭剂（BHQ）。 </li><li>进行定量PCR（定量聚合酶链式反应）中的25微升用340纳米的终体积反向ITR底漆，100nM的正向引物的ITR，100nM的AAV2 ITR探针，0.4毫的dNTPs，2mM的氯化镁，1个白金Taq缓冲液，1U白金Taq和5微升模板（标准，样品和无模板对照）。 </li><li>为的标准中，使用的质粒转染在7×10倍连续稀释液（每2×10 9个至2×10 3产生的最终浓度从10月10日至4月 10日的基因组/毫升的基因组/毫升5微升）。 </li><li>由初始变性步骤在95℃下进行15分钟，随后变性的40个循环的开始的程序，在95℃，1分钟退火/延伸60℃，1分钟。储存在4℃下短期储存，或在-20℃下长期储存。 </li></ol> 2.玻璃体内注射AAV的<ol><li>麻醉C57BL6J小鼠用氯胺酮（50毫克/千克）和赛拉嗪（10毫克/公斤），并验证翻正反射时，撤回反射和尾部夹送响应指示一个适当的麻醉的损失。由neosynephrine 5％的角膜应用扩张的学生和mydriaticum 0.5％眼药水。使用上的眼睛兽医药膏，以防止干燥，而在麻醉下。 </li><li>通过超细地下30 disposab乐针穿过赤道旁缘，入玻璃体腔（见邱K，朱庇特（2007）21）。注入1微升原料含有1至4×10 11 VP ShH10-GFP或AAV5-GFP与直接观察在玻璃体腔的中心针（ 图1）。用一只眼睛作为ILM实验对照。注入两只眼睛的BRB实验。对注入的眼睛敷上消炎和抗菌局部治疗。 </li><li>扩张与学生和neosynephrine mydriaticum眼药水成像。执行眼底检查与眼底照相机跟随的GFP（绿色荧光蛋白）的表达1周，2周，1个月，和玻璃体内注射后的2个月（ 图1）。用CO 2吸入1〜2个月后喷射牺牲小鼠。 </li></ol> 3.免疫组化<ol><li>视网膜的冷冻切片（ 图1），解剖剜出眼睛去除升在4％多聚甲醛1小时ENS和角膜，并浸泡修复。 <ol><li> Cryoprotect先前固定的眼睛在10％蔗糖1小时，在室温下，20％的蔗糖为另一小时，在室温下进行。 Cryoprotect在30％蔗糖溶液，过夜，在4℃。冻结和嵌入树脂如低温矩阵嵌入眼罩。使10微米的冰冻切片和安装在滑轨经过特殊处理的冷冻组织切片染色以及在改善组织粘连。 </li><li>透用0.1％的Triton X100的部分在PBS pH 7.4的5分钟。洗涤2次在PBS中，块1小时，在室温下在PBS中以1％BSA，0.1％吐温20。 </li></ol></li><li>视网膜flatmounts（ 图1），固定摘除眼睛用4％多聚甲醛中15分钟，以方便清扫。 <ol><li>在15-30分钟，解剖眼睛去除角膜和晶状体。通过切割周围锯齿缘分离的视网膜色素上皮细胞（RPE）和巩膜视网膜和视神经。沉浸在4％多聚甲醛另外30分钟来固定视网膜和在转导视网膜细胞的荧光蛋白（固定必须不超过24小时）。在无菌PBS，pH 7.4中洗涤5分钟。 </li><li>改变的PBS以封闭缓冲液（PBS中的1％BSA，2％正常山羊或驴血清，0.5％的Triton X-100），并孵育在封闭缓冲液为任一4小时，在室温下或4℃过夜。 </li></ol></li><li>孵育组织与初级抗体在封闭缓冲液为任一4小时，在室温下，或在4℃下过夜。洗涤3次，用PBS。 注意:是使用的抗体如下:抗层粘连蛋白（1 / 1,000）标记在ILM，抗视紫红质克隆4D2（1/500）标签棒，防谷氨酰胺合成克隆GS-6（1/1500）标签Müller细胞，PNA凝集素（1/40）标签锥。 </li><li>孵育组织用1:500稀释的Alexa Fluor缀合的第二抗体在封闭缓冲液中进行1小时（冷冻切片）或2小时（flatmounts），在室温坦佩叉涂抹并避光。洗涤3次，用PBS。 </li><li>使减轻削减视网膜和flatmount它与上无论是感光或视网膜神经节细胞（RGC）侧朝上载玻片。添加水mountingmedium，应用盖玻片并储存在4℃。 </li><li>执行激光扫描共聚焦显微镜共聚焦显微镜。由线获取图像顺序地，线，以减少激发和发射串扰。根据采样定理确定步长大小。使用曝光设置，最大限度地减少过饱和像素的最终图像。处理的12位图像斐济，项目的Z部分上使用下的Z-项目函数的最大强度并最终转换为8位的RGB色彩模式的单一平面。 </li></ol>小鼠血液样本4. PCR分析<ol><li>注入100μl的原料含有的AAV-GFP的1至4×10 11颗粒进入麻醉C57BL6J小鼠的阴茎静脉（异氟醚的5％用于使用胰岛素注射器用超细6毫米针诱导密切室，并通过在手术过程中一鼻锥异氟烷2.5％）。这种动物将作为循环的AAV颗粒的阳性对照。 </li><li>从鼠尾注射和3小时，24小时，2天，以及之后ShH10玻璃体内注射或intrapenile喷射（ 图1）后第3天前样品的血液。约10-20微升被收集在肝素管。在手术完成之后，牺牲小鼠通过CO 2吸入。 </li><li>提取使用你选择的提取试剂盒血液样本的基因组DNA。 </li><li>执行的基因组DNA的PCR扩增。此协议使用下列引物对:绿色荧光蛋白，有义:5&#39;-CGACACAATCTGCCCTTTCG-3&#39;，反义5&#39;-CATGGACGAGCTGTACAAGGGA-3&#39;。以下PCR程序进行:95℃2分钟（1个循环）; 95℃45秒，55℃1分钟，72℃45秒（30个循环）; 72°C 5分钟（1周期）;4℃保持。 </li></ol> 5.可选埃文斯蓝方法注:通过测量血管白蛋白漏入使用伊文思蓝法10视网膜量化血管通透性。 <ol><li>通过溶解在通过5μm过滤器的生理盐水溶液（6毫克/毫升），超声处理5分钟，在超声波洗涤机，并过滤制备伊文思蓝染料。 </li><li>麻醉C57BL6J小鼠（异氟烷吸入，见步骤4.1），检查翻正反射，撤退反射和尾掐响应，表示适当的麻醉的损失，并通过阴茎静脉（ 图1），注入伊文思蓝（45毫克/千克）。检查爪子，枪口，耳朵小鼠变得清楚蓝色以下注射Evans蓝染料，证实了染料的吸收和分布。 </li><li>注射用氯胺酮（50毫克/千克）和赛拉嗪（10毫克/千克）的染料后麻醉C57BL6J小鼠3小时，并验证rightin的损失克反射表明适当麻醉。样品心内约500微升的血液在肝素管，并通过左心室用柠檬酸盐缓冲液灌注小鼠，持续2分钟（0.05M，pH为3.5;溶解7.8克柠檬酸和3.8克柠檬酸钠在1升的蒸馏水中）预热至37℃，以防止血管收缩。小鼠经由灌注处死。 </li><li>后灌注，剜除，仔细解剖手术显微镜，收集视网膜下两只眼睛（见第3.2节）。在离心蒸发器中干燥视网膜过夜，称重，并通过培养视网膜用100μl甲酰胺中18小时，在65℃振荡（100 XG）提取伊文思蓝染料。在30K的ω过滤管在20,798.5 xg离心2小时，在4℃离心机视网膜样品离心血样在20,798.5 xg离心15分钟，在4℃。 </li><li>使用这两种上清测量吸光度。减去最大吸光度FO测定各样品的吸光度řEvans蓝在620nm到最小吸光度在740纳米。从伊文思蓝中甲酰胺的标准曲线计算出的伊文思蓝浓度在血浆和视网膜。表达BRB渗透性在每小时每克干视网膜（微升血浆/克视网膜干重/小时）的伊文思蓝的微升。 </li></ol> 6.可选荧光血管造影注:泄漏从视网膜血管小鼠可通过腹膜内注射荧光素钠被可视化。 <ol><li>麻醉C57BL6J小鼠（异氟烷吸入，见步骤4.1）和瞳孔扩张与眼药水（neosynephrine和mydriaticum）。使用上的眼睛兽医药膏，以防止干燥，而在麻醉下。 </li><li>注入腹膜内0.01-0.02毫升10％荧光素钠在0.1ml无菌盐水进行荧光血管造影。 </li><li>收集双眼用眼底照相机注射后的图像。后亲的CO牺牲小鼠吸入2cedure。 注:20秒所必需的视网膜血管，以成为对造影可见。图片必须要3-10分钟最大荧光素注射后抓获。漏点（如果存在）2.5分钟后可观察到。在稍后的时间点的图像质量由于荧光素钠扩散到用于分析之后不久得到的玻璃体，因此仅图像丢失。 </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+adeno-associated+virus-mediated+gene+delivery+in+retinal+degeneration.">Changes in adeno-associated virus-mediated gene delivery in retinal degeneration.</a> Hum Gene Ther. 21 (5), 571-578 (2010).</li><li>Sene, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+implication+of+Dp71+in+osmoregulation+and+vascular+permeability+of+the+retina.">Functional implication of Dp71 in osmoregulation and vascular permeability of the retina.</a> PLoS One. 4 (10), e7329 (2009).</li><li>Benard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Quantifiable+Blood+Retinal+Barrier+Breakdown+Model+In+Mice.">A New Quantifiable Blood Retinal Barrier Breakdown Model In Mice.</a> ARVO Annual Meeting. , (2011).</li></ol>

The authors have nothing to disclose.

该BRB调节血液和视网膜之间的分子交换。其击穿与各种疾病，如糖尿病性视网膜病或年龄相关性黄斑变性（AMD）相关联。我们最近发现，在一个肌营养不良蛋白敲除小鼠，其中显示可渗透BRB，视网膜变得更宽容的由腺相关病毒载体（AAV）介导的基因传递。然而，尽管BRB渗透性的AAV颗粒喷入眼内保持局限于此模型中的眼隔室。我们的研究结果表明，基因治疗中显示渗透BRB并不代表全身副作用附加险?...

Müller细胞是视网膜的主要神经胶质成分。形态，它们跨越视网膜径向和它们endfeet，与玻璃体接触，面对在ILM和后者的秘密分量。在ILM是一种基底膜约十个不同的细胞外基质蛋白（层粘连蛋白，聚集蛋白，串珠素，巢蛋白，胶原蛋白和一些硫酸肝素蛋白聚糖）组成。在开发过程中，它的存在是不可或缺的视网膜组织发生神经节细胞1-3，视神经轴突的导航和生存。但是，ILM是非本质的成人视网膜，可以在某些病理手术切除，而不会造成视网膜损伤4。在基因治疗中，此膜变成为使用自动增值服务的视网膜高效转导通过玻璃体内注射5的物理屏障。 通过他们的流程的广泛树枝状，Müller细胞提供营养和监管suppoRT既视网膜神经细胞和血管细胞。 Müller细胞也参与视网膜动态平衡的调节，在BRB 6的形成和维持。紧密连接视网膜毛细血管内皮细胞之间，Müller细胞，星形胶质细胞和周形成BRB。 BRB防止某些物质进入retina.In许多疾病，如糖 ​​尿病性视网膜病变，视网膜静脉阻塞和呼吸系统疾病，视网膜缺氧会导致通过BRB 7-9泄漏。这种断裂是与增加血管通透性，导致血管性水肿，视网膜脱离和视网膜损伤相关联。 Müller细胞被紧密地与血管和基底膜相关联，打在两BRB和ILM完整性中起重要作用。因此，标记缪勒胶质细胞是特别相关的这些视网膜屏障的物理状态的研究。 经典同盟，BRB渗透性是用伊文思蓝测定法，包括全身性注射Evans蓝染料，其结合非共价地与血浆白蛋白的测定。此法测量从血管进入视网膜（见协议第5条）10白蛋白漏出（蛋白质中等大小，〜66 kDa的）。另外，血管渗漏可以通过荧光血管造影视网膜的证明荧光素渗漏进行可视化（小分子〜359达;见协议第6条）11。然而，这两种方法允许BRB渗透性小分子和蛋白质的评估，但它们不提供关于在ILM的完整性信息。 因此，为了研究BRB渗透性，我们使用的AAV基础的方法，让上BRB渗透性至较大的分子（ 例如，AAV颗粒，25纳米的直径）的信息。事实上，我们的方法可以检测出的AAV转基因的存在于血液中，这将表明，〜25 nm的直径的颗粒会能够渗透到血液中。这种方法还提供了在病理条件下在ILM和细胞外基质蛋白质的结构信息。两个变种AAV对于这样的研究有用:AAV5和ShH10。视网膜下注射，AAV5具有天然向性为感光细胞和视网膜色素上皮细胞12，但是它不能获得跨越到外视网膜给药时进入玻璃体在野生型视网膜中有完整的ILM 5,13。 ShH10是已工程化以特异性靶向神经胶质细胞在神经元14,15的AAV变种。 ShH10选择性标记在健康和患病的视网膜与视网膜增加的效率受损障碍16Müller细胞。再加immuhistochemistry和血液DNA分析这些病毒的工具提供的视网膜屏障的状态和它们在疾病的参与（ 图1）的信息。

<table><tbody><tr><td>C57BL6J mice strain</td><td>JANVIER LABS</td><td></td><td>mice</td></tr><tr><td>Ketamine 500</td><td>Virbac France</td><td></td><td>anesthetic</td></tr><tr><td>Xylazine Rompun 2%</td><td>Bayer Healthcare</td><td></td><td>anesthetic</td></tr><tr><td>Neosynephrine 5% Faure</td><td>Europhta</td><td></td><td>dilatant</td></tr><tr><td>Mydriaticum 0.5%</td><td>Thea</td><td></td><td>dilatant</td></tr><tr><td>Sterdex</td><td>Novartis</td><td></td><td>anti-inflammatory</td></tr><tr><td>Cryomatrix embedding resin</td><td>Thermo Scientific</td><td>6769006</td><td></td></tr><tr><td>Superfrost Plus Adhesion Slides</td><td>Thermo Scientific</td><td>10143352</td><td>slides</td></tr><tr><td>anti-laminin</td><td>Sigma</td><td>L9393</td><td>antibody</td></tr><tr><td>anti-rhodopsin clone 4D2</td><td>Millipore</td><td>MABN15</td><td>antibody</td></tr><tr><td>anti-glutamine synthetase clone GS-6</td><td>Millipore</td><td>MAB302</td><td>antibody</td></tr><tr><td>Anti-Glial Fibrillary Acidic Protein</td><td>Dako</td><td>334</td><td>antibody</td></tr><tr><td>PNA Lectin</td><td>Invitrogen</td><td>L32459</td><td>probe</td></tr><tr><td>Alexa fluor conjugated secondary antibodies</td><td>Invitrogen</td><td></td><td>antibody</td></tr><tr><td>Fluorsave reagent</td><td>Calbiochem</td><td>345789</td><td>mounting medium</td></tr><tr><td>QIAmp DNA Micro Kit</td><td>QIAGEN</td><td>56304</td><td></td></tr><tr><td>GoTaq DNA polymerase</td><td>Promega</td><td>M3001</td><td></td></tr><tr><td>Evans Blue dye</td><td>Sigma</td><td>E2129</td><td>dye</td></tr><tr><td>5 &amp;micro;m filter</td><td>Millipore</td><td></td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S1804</td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C1909-2.5KG</td><td></td></tr><tr><td>Formamide spectrophotometric</td><td>Sigma</td><td>295876-2L</td><td></td></tr><tr><td>Fluorescein</td><td>Sigma</td><td>F2456</td><td>dye</td></tr><tr><td>Micron III</td><td>Phoenix Research Labs</td><td></td><td>Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.</td></tr><tr><td>Insulin Syringes</td><td>Terumo</td><td>SS30M3109</td><td></td></tr><tr><td>Syringe 10 &amp;micro;l Hamilton</td><td>Dutscher</td><td>74487</td><td>Seringue 1701</td></tr><tr><td>Needle RN G33, 25 mm, PST 2</td><td>Fisher Scientific</td><td>11530332</td><td>Intravitreal Injection</td></tr><tr><td>UltraMicroPump UMP3</td><td>World Precision Instruments</td><td>UMP3</td><td>Versatile injector uses microsyringes to deliver picoliter volumes</td></tr><tr><td>UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller</td><td>UMP3-1</td><td>Digital controller</td><td></td></tr><tr><td>Binocular magnifier SZ76</td><td>ADVILAB</td><td>ADV-76B2</td><td>Zoom 0.66 x 5 x LEDs with stand epi and dia / Retinas dissection</td></tr><tr><td>Spring scissors straight - 8.5 cm</td><td>Bionic France S.a.r.l</td><td>15003-08</td><td>Retinas dissection</td></tr><tr><td>curved Vanna scissor</td><td>15004-08</td><td></td><td></td></tr><tr><td>Pince Dumont 5</td><td>11254-20</td><td></td><td></td></tr><tr><td>Veriti 96-Well Thermal Cycler</td><td>Life technologies</td><td>4375786</td><td>Thermocycler</td></tr><tr><td>Ultrasonic cleaner</td><td>Laboratory Supplies</td><td>G1125P1T</td><td></td></tr><tr><td>Nanosep 30k omega tubes</td><td>VWR</td><td></td><td></td></tr><tr><td>Speedvac</td><td>Fisher Scientific</td><td>SC 110 A</td><td></td></tr><tr><td>Spectrofluorometer</td><td>TECAN</td><td>infinite M1000</td><td></td></tr></tbody></table>

using adeno-associated virus as a tool to study retinal barriers in disease

Müller细胞是视网膜的主要神经胶质细胞。其最终的脚形成的视网膜在外侧和内界膜（ILM）的范围内，并在与星形胶质细胞，周细胞和内皮细胞结合它们建立血 - 视网膜屏障（BRB）。 BRB限制了血液和视网膜之间的物质运输而在ILM充当基底膜限定组织学视网膜和玻璃体腔之间的边界。标记Müller细胞是特别相关，研究视网膜屏障的物理状态，因为这些细胞是BRB和ILM的一个组成部分。既BRB和ILM经常改变视网膜病和负责疾病症状。 有几种公认的方法来研究的BRB的完整性，如Evans蓝测定法或荧光血管造影。然而，这些方法不提供关于BRB渗透性吨的范围内的信息Ø较大的分子，在纳米范围内。而且，它们不提供对其他视网膜屏障如在ILM的状态信息。研究BRB渗透性沿着视网膜ILM，我们使用的AAV基础的方法，可提供对渗透性BRB对较大分子虽然表示在疾病状态在ILM和胞外基质蛋白的状态的信息。两个变种AAV对于这样的研究有用:AAV5和ShH10。 AAV5具有天然趋光感受器，但它不能得到跨越到外视网膜给药时进入玻璃体时在ILM是完整的（ 即，在野生型视网膜）。 ShH10具有朝向胶质细胞强烈的取向和在健康和患病的视网膜将选择性标记米勒胶质细胞。 ShH10提供视网膜更高效的基因传递，其中ILM受到损害。再加上免疫和血液DNA分析这些病毒工具揭示到的视网膜疾病障碍的状态。

我们预计增加的使用ShH10缪勒胶质细胞的视网膜转导，如果该动物模型显示扰动在ILM（ 图2A - B）的结构。例如，我们已经表明，在不存在的Dp71，ShH10靶特异性而更有效地缪勒胶质细胞通过玻璃体内注射，这表明在ILM的通透性增加在该鼠标线相比野生型小鼠16（ 图2C - F）。 AAV5也可以用作ILM渗透性的指标。 AAV5，无效通...

Watch this Scientific Journal Video about Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease at JoVE.com

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

using-adeno-associated-virus-as-tool-to-study-retinal-barriers

Research

JoVE Journal

Medicine

15.0K 次观看.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

视频: Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

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.

病毒遗传背景明确的神经回路跟踪

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

科研

神经科学

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.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

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 ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

医学

通过单步肝素亲和层析纯化 AAV 载体

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

作者聚焦：腺相关病毒载体生产和纯化的改进方法

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

生物工程

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

数字液滴PCR法定量分析小鼠视网膜AAV转导效率

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

小动物模型中腺相关病毒载体的结膜下给药

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

AAV 滴定定量斑点印迹法及其在腺相关病毒组装活化蛋白功能评价中的应用

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

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病毒遗传背景明确的神经回路跟踪

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

数字液滴PCR法定量分析小鼠视网膜AAV转导效率

小动物模型中腺相关病毒载体的结膜下给药

AAV 滴定定量斑点印迹法及其在腺相关病毒组装活化蛋白功能评价中的应用