Name: methods and tips for intravenous administration of adeno-associated virus to rats and evaluation of central nervous system transduction
Uploaded: 2017-08-25T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction at JoVE.com

这项工作由 als 协会, Karyopharm 疗法, Inc., Meira GTx, 和一个慈善捐赠的 als 研究从托马斯劳森, 我们很感激。我们感谢 Elysse 果园和唐娜伯尼的建议和培训。JAS 得到了撒谎基金会和国立卫生研究院 GM103418 的支持。

所有程序都遵循了适当的 NIH 指南。所有的程序都遵循动物协议, 这是由位于什里夫波特的路易斯安那州立大学健康科学中心的动物保育和使用委员会批准的。6周龄的雌性大鼠被用于此手术. 
 1. 确定所需的向量的剂量/体积 <ol> <li> 将被注射的老鼠称重, 并根据载体制剂的滴度 (浓度) 确定每种动物所需的病毒载体的数量。使用的矢量剂量已经成功地在大鼠中枢神经系统的表达之前 (见杰克逊 et al . 6 ). 注意: 对于 AAV9 TDP-43 或 AAV9 GFP, 典型剂量介于 3 x 10 13 -1 x 10 14 矢量基因组/千克之间。6周龄的成年小鼠体重约150克. </li>
	<li> 在所有动物中每千克当量剂量进行系统比较。确保音量不超过标准建议的注塑量 (250-500 和 #181; L 为250克鼠)。一个典型的注射量是200和 #956; 我对一个年轻的成年老鼠. </li>
</ol> 2。准备工作站 <ol> <li> 收集所有必需的耗材: 每只动物一个石蜡薄膜正方形 (约 5 x 5 厘米), 每只动物一个酒精制剂垫, 纱布垫 (每只动物 2-3), 吸管提示和微. </li>
	<li> 用70% 乙醇清洗表面, 准备工作表面。将加热垫放下, 温度设置为低 (〜37和 #176; C)。将一个长凳垫放在暖气垫的顶部. </li>
	<li> 准备气体麻醉 (异氟醚)。确保充分的氧气和气体麻醉的程序。用70% 乙醇清洗感应室和麻醉面罩, 并将麻醉面罩放在工作台垫上. </li>
</ol> 3。准备矢量 <ol> <li> 在室温下解冻向量, 并为每个动物标记一个无菌离心管. </li>
	<li> 吸管将步骤1.1 中确定的 AAV 的体积转换成适当的管, 并使音量达到所需的注入量 (这里, 200 和 #956; l) 与林格林格和 #39; s 溶液或盐水. </li>
	<li> 脉冲涡流样品 1-2 s, 然后脉冲离心机 5 s, 使样品到底部的管. </li>
	<li> 吸管的总注射量为鼠出管和到广场的石蜡薄膜. </li>
	<li> 在1毫升注射器上放置一个30口径的针. </li>
	<li> 将针与斜面朝下放置在石蜡膜上的矢量上。拉回注射器柱塞, 直到所有的病毒是在针头和注射器。小心避免把气泡拉进针里, 这样练习就容易多了. </li>
</ol> 4。准备大鼠 <ol> <li> 将氧气升至1升/分, 并将异氟醚麻醉设置为5%。通过检查所有连接软管和塞方向, 确保气体麻醉流向感应室. </li>
	<li> 将大鼠置于气体麻醉诱导室中约3分钟, 直到老鼠没有反应. </li>
	<li> 通过捏脚趾来确保充分的麻醉深度。脚趾捏后, 脚或腿不应有运动. </li>
	<li> 将异氟醚麻醉减少到 2%, 用于麻醉的维护, 调整塞, 使麻醉流到麻醉面罩上. </li>
	<li> 将老鼠和 #39 的鼻子放在麻醉面罩里, 调整老鼠的位置, 使它躺在它的侧面. </li>
	<li> 识别侧尾静脉. 
	注: 侧尾静脉位于大约10和 2 o 和 #39; 时钟与尾部的背顶部作为 12 o 和 #39; 时钟。当老鼠躺在它的一侧时, 侧尾静脉应该朝上. </li>
	<li> 用酒精准备垫擦拭注射区域。注射地点是2/3 下长度的尾巴. </li>
</ol> 5。注入矢量 <ol> <li> 将尾部略微固定在注射区域上方, 用一根手指直接越过侧尾静脉; 这会放大静脉. </li>
	<li> 将斜面朝上, 将针与可见尾静脉对准同一方向. </li>
	<li> 在非常小心的时候, 在尾部静脉上刺穿皮肤, 避免另一只手按住尾部并按下尾部静脉. </li>
	<li> 释放来自尾静脉的压力以允许正常的血流. </li>
	<li> 将针头移入静脉。为了确保针头刺穿了尾静脉, 在柱塞上稍微向后拉。如果针头在静脉, 血液就会流入针头。
	<ol> <li> 根据需要重新定位针。然后, 为了稳定针头, 将手移到注射部位的下方, 并将注射器的末端固定在尾部. </li>
	</ol> </li> <li> 将向量慢慢注入尾部 (大约20和 #181;
	注: 当矢量流经时, 静脉可能会失去颜色。在静脉外注入溶液的迹象包括起泡, 皮肤的烫漂, 或注射载体时的阻力。通过实践, extra-vascular 的曝光率可以最小化. </li>
	<li> 在该载体被管理后, 从鼠身上取出针头, 并在注射部位上压上纱布垫以帮助 30-六十年代的出血. </li>
</ol> 6。清除 <ol> <li> 关闭异氟醚麻醉和氧气。监测鼠, 直到它恢复知觉, 然后返回到它的笼子. </li>
	<li> 小心地把所有的针头放在锋利的容器里。将可能被 AAV 污染的所有其他一次性材料处理成适当的生物危害容器。用70% 乙醇清洁工作站. </li>
</ol> 7。评估后肢逃生反射 注: 在 TDP-43 基因转移后, 后肢缺损通常出现 2-6 周, 这取决于载体剂量. 
<ol> <li> 在平坦的表面上清除碎片, 用两个手指抓住鼠和 #39; 尾巴大约2厘米从身体。轻轻抬起大鼠, 直到前肢在观察鼠腹底时悬挂。
	<ol> <li> 在基因转移 (例如, 基因转移后12周) 的整个实验时效中每周执行过程. 
		注: 也以这种方式得分前肢赤字, 但后肢赤字更有可能发生在这个模型, 取决于所使用的向量剂量. </li>
	</ol> </li> <li> 观察十年代的后肢. 注意, 如果一个或两个后肢紧握对中线在十年代. </li>
	<li> 重复此测试总共三次, 与三十年代之间的试验。如果一个或两个后肢是紧握的每三试验, 老鼠显示运动功能障碍。记录数据 (手动在笔记中), 无论是单边或双边后肢赤字存在, 如果有一致的紧握在所有三试验. </li>
</ol> 8。在 rotarod 上评估马达功能 <ol> <li> 设置 rotarod, 在 rotarod 下放置一个工作台垫, 并将加速度设置为每分钟 4-40 转 (rpm) 超过2分钟 (速率增加了 ~ 0.3 rpm/秒)。12周的时效, 在基因转移后的2、4、8和12周内进行实验. </li>
	<li> 允许老鼠适应测试室20分钟. </li>
	<li> 训练大鼠第一次使用 rotarod, 首先以 4 rpm 的速度启动 rotarod, 然后将老鼠放在 rotarod 上。允许老鼠在4分钟的转速下进行一次全面的革命, 然后开始加速, 让老鼠在 rotarod 上行走, 直到它脱落。继续训练, 直到老鼠能持续至少四十年代的步行。一个健康的, 年轻的成年鼠将容易接受这种方式的训练。
	<ol> <li> 在有明显的运动损伤的大鼠的情况下, 这可能会阻止大鼠在四十年代底之前达到基线潜伏期, 在开始 rotarod 评分之前提供 3-5 的训练尝试. </li>
	</ol> </li> <li> 开始 rotarod 测试, 将鼠放在 rotarod 上, 并开始加速 rotarod。注意老鼠从 rotarod 上掉下的时间;这是下降的潜伏期。如果老鼠仍然在 rotarod 在120s 以后, 盖帽会议在这一点上, 去除鼠从 rotarod 并且记录秋天潜伏期 120s. </li>
	<li> 对总共三项试验重复测试两次。为了尽量减少疲劳的影响, 试验间隔至少5分钟。平均三项试验的得分。未受损伤的成年鼠通常有平均潜伏期和 #62; 四十年代. </li>
	<li> 将老鼠放回笼子里, 用70% 乙醇清洁 rotarod 和周围的区域. </li>
</ol> 9。在小脑中荧光蛋白表达的量化 <ol> <li> 麻醉鼠和灌注与磷酸盐缓冲盐水 (PBS), 然后4% 甲醛。解剖大脑和脊髓, 并浸泡在定影液过夜, 然后将其移动到30% 蔗糖溶液。平衡后, 削减50和 #956; m 冠状部分在滑动切片与冻结阶段 4 , 5 , 6 。
	<li> 选择小脑的3-6 节, 在小脑中央裂片的蚓中均匀分布。荧光标签的 GFP, 以最大限度的信号。使用 GFP 的主要抗体在1:500 和使用 Alexa-488 共轭二级抗体在稀释 1:300 4 , 5 , 6 。
	注意: 有关取样、切片和染色过程的详细信息, 请参阅 4 、 5 、 6 。
	<li> 显微使用显微镜对所定义的感兴趣区域中的每个剖面进行低放大透镜。使用2.5X 目标 (0.12) 和过滤器设置 10, 450-490/515-565 的励磁/发射)。保持相机设置 (曝光时间, 例如, 2 秒) 相同的跨样品进行分析。另存为。TIF 文件. </li>
	<li> 在广泛可用的接穗图像程序中打开显微; 将打开两个窗口。关闭指示为和 #34 的窗口; 索引颜色和 #34;. </li>
	<li> 选择和 #34; 选项和 #34;, 然后 #34;D ensity 切片和 #34;; 在 "查找表" 窗口中, 将用红色表示一系列色调。在窗口中, 使用光标只填充特定的 GFP 标签, 并在图像中开始拾取非特异背景时停止. 
	注: 这将突出显微上的荧光区域将被量化。这些设置应进行优化, 以专门捕获所有可见的荧光细胞。实践表明, 该方法能准确地突出特定的荧光特性. </li>
	<li> 在测量荧光区域之前, 首先要确保所做的测量是正确的单位 (像素)。为此, 请单击并 #34; 分析和 #34;, 然后 #34; 设置规模和 #34;;第四行应该说和 #34; 单位和 #34;。从下拉列表中, 选择像素以像素为单位, 然后单击并 #34; 好的 #34。将分析选项切换到和 #34; 包括内部孔和 #34; 确保在分析中包含整个单元体. </li>
	<li> 测量荧光区域, 单击和 #34; 分析和 #34; 然后单击和 #34; 测量和 #34;。要查看此值, 请单击 "#34"; 分析和 #34; 然后单击并 #34; 显示结果和 #34;;将出现一个结果窗口。荧光区域测量将在标题和 #34; 面积和 #34;. </li>
	<li> 若要确保整个测量的标准化, 请不要更改 "查找" 窗口中突出显示的值. </li>
	<li> 一旦记录了所有的测量值, 每个动物的平均值 (每只动物的 3-6 节)。使用适当的统计测试来比较组之间的转区域。 6 </li> </ol>

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作者没有什么可透露的。

各种分娩路线已测试 AAV 载体: intra-parenchymal, intra-cerebroventricular, intra-thecal, 静脉注射, 鼻腔, intra-muscular 等。每条路线可能有具体的优点和缺点。尾静脉 AAV 对成年大鼠是一种可靠的方法, 以实现一致转导的中枢神经系统。外周注射法避免将注射套管引入中枢神经系统组织, 因此是微创的。这种方法能实现的有效的 CNS 表达是该技术的最大优势。其他的实际优势是, 注射法是非常快 (约10分钟每动物从?...

重组腺相关病毒 (AAV) 载体是中枢神经系统研究不可缺少的工具, 因为它们对换神经元的体内有效。AAV 载体对于研究不同的转基因和蛋白质异构体、不同的组织、不同的寄主种类和不同的管理途径是非常通用的。例如, AAV 可以通过外周、相对非侵入性、静脉注射到中枢神经系统的传感器神经元来进行小鼠的管理, 这首先在 Foust et al.和杜克et al.中描述(参见朱庇特纸由 Gombash et al.(2014))。1,2,3这一基因传递方法用于大鼠有效表达绿色荧光蛋白 (GFP) 或 ALS 相关蛋白、交互反应 DNA 结合蛋白 43 kDa (TDP-43) 的中枢神经系统。4,5,6,7,8,9在大鼠中工作是很重要的, 因为老鼠的生理和代谢参数与小鼠相比更接近人类, 而且还有专门为老鼠设计的行为和毒理学化验。此外, 越来越多的转基因大鼠品系可用于 AAV 基因转移研究。
方法是详细的大鼠扩张 CNS 基因转移, 并迅速, 可靠的量化结果。广泛的中枢神经系统转导是用来模拟的症状性 ALS 在大鼠通过表达 TDP-43 整个脊髓。该方法是在杰克逊et al.中使用的 TDP-43 载体的尾静脉注射。6,8,9在几周后, TDP-43-induced 的马达缺陷分为两种方法: 转义反射和 rotarod, 用于代顿et al.和杰克逊et al.5,6,7,9为控制 GFP 载体, 在验尸分析中, 小脑的萤光区被计算为在杰克逊et al.中使用的转导效率指数。6,8小脑的分析已被证明是一个快速和可靠的指标的程度中枢神经系统转导后, 外周基因交付, 并应适用于各种方法试图 peripheral-to-central 基因转移。

<table><tbody><tr><td>Scale</td><td>Pelouze</td><td>SP5</td><td></td></tr><tr><td>Nalgene bucket</td><td>Thermo Scientific</td><td>12014000</td><td>4000 mL</td></tr><tr><td>Microcentrifuge tube</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>Lactated Ringer&#039;s Solution</td><td>Baxter Healthcare</td><td>0338-0114-04</td><td></td></tr><tr><td>Mini vortexer</td><td>Fisher Scientific</td><td>128101</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>22624415</td><td></td></tr><tr><td>Laboratory film</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>1-mL syringe</td><td>BD</td><td>309626</td><td>Comes with a 25g needle attached.&lt;br/&gt; Replace with 27-30g needle for injection</td></tr><tr><td>30g needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Healthcare</td><td>66794-013-25</td><td>Isoflurane USP 250 mL</td></tr><tr><td>Anesthesia mask</td><td>Kopf Instruments</td><td>906</td><td></td></tr><tr><td>Gauze</td><td>Henry Schein</td><td>100-2524</td><td>2&quot; x 2&quot; (5cm x 5cm)</td></tr><tr><td>Pipet tips</td><td>USA Scientific</td><td>1111-0816</td><td></td></tr><tr><td>Micropipet</td><td>Gilson</td><td>4642080</td><td></td></tr><tr><td>Gas anesthesia vaporizer</td><td>SurgiVet</td><td>4214322</td><td>Classic T3</td></tr><tr><td>Gas anesthesia scavenger</td><td>SurgiVet</td><td>32373B10</td><td>Enviro-PURE</td></tr><tr><td>Heating pad</td><td>Sears</td><td>17280</td><td></td></tr><tr><td>Bench pad</td><td>VWR</td><td>56617-006</td><td></td></tr><tr><td>Rotarod</td><td>Panlab/Harvard Apparatus</td><td>76-0772</td><td>4 rats/ 4 mice</td></tr><tr><td>70% Ethanol</td><td>Decon Laboratories, Inc.</td><td>2401</td><td>140 proof</td></tr><tr><td>70% Isopropanol</td><td>VWR</td><td>89108-160</td><td></td></tr><tr><td>Scion Image</td><td>Scion Corporation</td><td></td><td></td></tr><tr><td>GFP Tag Polyclonal Antibody</td><td>Invitrogen</td><td>A11122</td><td>Primary Antibody - 1:500 dilution</td></tr><tr><td>AlexaFluor 488</td><td>Invitrogen</td><td>A11070</td><td>Secondary Antibody - 1:300 dilution</td></tr><tr><td>Axioskop 40 microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

methods and tips for intravenous administration of adeno-associated virus to rats and evaluation of central nervous system transduction

腺相关病毒 (AAV) 载体是神经中的一个关键试剂, 用于 interspaced 短复发重复 (CRISPR)、遗传学、液氧靶向等。这份手稿的目的是帮助研究者尝试通过尾静脉注射 AAV 大鼠的扩张中枢神经系统 (CNS) 基因转移。广泛的表达是相关的条件与广泛的病理, 和大鼠模型是重要的, 因为它的大小和生理上的相似性, 与人类相比, 小鼠。在这个例子应用中, 一个广泛的神经传导是用来模拟一种神经退行性疾病, 影响整个脊髓, 肌萎缩侧索硬化症 (ALS)。有效的广泛的中枢神经系统转导也可以用于提供临床前研究中的治疗蛋白因子。经过数周的后表达间隔后, 对转导的影响进行了评价。对于绿色荧光蛋白 (gfp) 控制向量, 在小脑中 GFP 的数量是由一个基本的成像程序快速和可靠的估计。对于由 ALS 相关蛋白交互应答 DNA 结合蛋白诱导的 43 kDa (TDP-43) 的运动疾病表型, 通过逃逸反射和 rotarod 来评分。除了疾病建模和基因治疗, 在这里描述的广泛的基因打靶有不同的潜在应用。扩大使用这种方法将有助于加速假设测试在神经和遗传学。

TDP-43 引起的运动损伤应在 2-6 周内出现, 这取决于使用的 AAV TDP-43 的剂量 (图 1)。不成功的尾静脉注射会导致局部或无损伤;AAV 必须到达血流才能产生效果。成功注射 AAV gfp, 将导致在整个大脑和脊髓的 gfp 表达, 以向量剂量的依赖方式。当使用强的重组启动子驱动表达, 巨细胞病毒鸡β-肌动蛋白混合启动子, 也称为 CAG 启动子, 有有效的表达在中枢神经系...

Watch this Scientific Journal Video about Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction at JoVE.com

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

方法为广泛的中枢神经系统基因交付在大鼠被盖。在这个例子中, 目的是模拟一种疾病, 影响整个脊髓。广泛的转导可以用来提供一个治疗蛋白的中枢神经系统从一个 one-time, 外围管理。

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

methods-tips-for-intravenous-administration-adeno-associated-virus-to

Research

JoVE Journal

Neuroscience

11.5K 次观看.  Louisiana State University Health Sciences Center. 方法为广泛的中枢神经系统基因交付在大鼠被盖。在这个例子中, 目的是模拟一种疾病, 影响整个脊髓。广泛的转导可以用来提供一个治疗蛋白的中枢神经系统从一个 one-time, 外围管理。

视频: Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

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.

颅内注射腺相关病毒载体

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

科研

神经科学

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.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

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

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

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 (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of 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 ...

医学

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

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.

在成年小鼠中的亚型腺相关病毒9（AAV9）载体递送

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.

腺相关病毒介导的脊髓基因神经元的转基因表达

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.

重组腺相关病毒在成年小鼠中的直接注射

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.

与腺相关病毒的向量的生产、纯化和质量控制

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

幼年大鼠腰椎穿刺 AAV9 递送用于 CNS 基因治疗

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.

作者聚焦：评估幼年大鼠的鞘内基因治疗疗效

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.

通过腰椎池注射在幼年大鼠中鞘内载体给药

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 ...

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

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此视频中的章节

颅内注射腺相关病毒载体

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

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

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

在成年小鼠中的亚型腺相关病毒9（AAV9）载体递送

腺相关病毒介导的脊髓基因神经元的转基因表达

重组腺相关病毒在成年小鼠中的直接注射

与腺相关病毒的向量的生产、纯化和质量控制

通过腰椎池注射在幼年大鼠中鞘内载体给药

通过腰椎池注射在幼年大鼠中鞘内载体给药