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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Ther. 17 (7), 1187-1196 (2009).</li><li>Gombash Lampe, S., Kaspar, B., Foust, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+injections+in+neonatal+mice.">Intravenous injections in neonatal mice.</a> J. Vis. Exp. (93), e52037 (2014).</li><li>Wang, 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=Expansive+gene+transfer+in+the+rat+CNS+rapidly+produces+amyotrophic+lateral+sclerosis+relevant+sequelae+when+TDP-43+is+overexpressed.">Expansive gene transfer in the rat CNS rapidly produces amyotrophic lateral sclerosis relevant sequelae when TDP-43 is overexpressed.</a> Mol. 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Physiol. Funct. Imaging. 23 (5), 237-246 (2003).</li></ol>

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&#39;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. 
			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&#34; x 2&#34; (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

11.4K 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

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

在小鼠模型中静脉自我管理的方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

生物神经毒素在茎的网络文化功能评估细胞衍生的中枢神经系统神经元

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

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. ...

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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