Name: subpial adeno-associated virus 9 (aav9) vector delivery in adult mice
Uploaded: 2017-07-13T12:30:00
Description: Watch this Scientific Journal Video about Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice at JoVE.com

这项研究得到了SANPORC和ALSA基金会赠款（Martin Marsala）的支持。国家可持续发展计划，项目编号LO1609（捷克教育，青年和体育部）;和RVO：67985904（Stefan Juhas和Jana Juhasova）。

这些研究是根据加利福尼亚大学圣地亚哥分校动物保健和使用委员会批准的方案进行的，并符合动物实验动物保护评估指南。所有研究都以使组大小和动物痛苦最小化的方式进行。 一般动物和手术准备<ol><li> 开始外科手术之前，解冻病毒（AAV9-UBI-GFP;5μL等分试样） 8 。通过将葡聚糖粉末在蒸馏水中混合，制备5％葡聚糖（10,000 MW）溶液。将病毒溶液与1...

<ol>
	<li>Foust, K. 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=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat Biotechnol. 27 (1), 59-65 (2009).</li><li>Duque, S., 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=Intravenous+administration+of+self-complementary+AAV9+enables+transgene+delivery+to+adult+motor+neurons.">Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.</a> Mol Ther. 17 (7), 1187-1196 (2009).</li><li>Gray, S. J., 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=Preclinical+differences+of+intravascular+AAV9+delivery+to+neurons+and+glia:+a+comparative+study+of+adult+mice+and+nonhuman+primates.">Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates.</a> Mol Ther. 19 (6), 1058-1069 (2011).</li><li>Meyer, K., 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=Improving+single+injection+CSF+delivery+of+AAV9-mediated+gene+therapy+for+SMA:+a+dose-response+study+in+mice+and+nonhuman+primates.">Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates.</a> Mol Ther. 23 (3), 477-487 (2015).</li><li>Foust, K. 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=Therapeutic+AAV9-mediated+suppression+of+mutant+SOD1+slows+disease+progression+and+extends+survival+in+models+of+inherited+ALS.">Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.</a> Mol Ther. 21 (12), 2148-2159 (2013).</li><li>Passini, M. 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=Translational+fidelity+of+intrathecal+delivery+of+self-complementary+AAV9-survival+motor+neuron+1+for+spinal+muscular+atrophy.">Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.</a> Hum Gene Ther. 25 (7), 619-630 (2014).</li><li>Bell, P., 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=Motor+neuron+transduction+after+intracisternal+delivery+of+AAV9+in+a+cynomolgus+macaque.">Motor neuron transduction after intracisternal delivery of AAV9 in a cynomolgus macaque.</a> Hum Gene Ther Methods. 26 (2), 43-44 (2015).</li><li>Miyanohara, 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=Potent+spinal+parenchymal+AAV9-mediated+gene+delivery+by+subpial+injection+in+adult+rats+and+pigs.">Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.</a> Mol Ther Methods Clin Dev. 3, 16046 (2016).</li><li>Xu, Q., 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=In+vivo+gene+knockdown+in+rat+dorsal+root+ganglia+mediated+by+self-complementary+adeno-associated+virus+serotype+5+following+intrathecal+delivery.">In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.</a> PLoS One. 7 (3), 32581 (2012).</li><li>Xiao, X., Li, J., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+recombinant+adeno-associated+virus+vectors+in+the+absence+of+helper+adenovirus.">Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.</a> J Virol. 72 (3), 2224-2232 (1998).</li></ol>

目前的研究描述了一种在成年小鼠中的辅助载体（AAV9）递送技术。如所附视频所示，该方法和技术可以有效地被使用，只要根据既定和测试的规格，正确地制造所需的仪器和穿刺针和腋下注射针。 在小鼠中进行一致和安全的唾液注射的关键技术变量： 如所证明的，在背侧颈椎或腰椎板切除术后，可以在成年麻醉的小鼠中容易地进行支气管注射。有几个关键...

使用AAV载体治疗各种脊髓和CNS神经退行性疾病正在成为一个被广泛接受的平台，可有效地上调或沉默感兴趣的基因的表达。更有效地利用该技术治疗CNS /脊髓障碍的关键限制之一是将AAV载体递送到成年哺乳动物的深部脑或脊髓实质的能力有限。 据证实，例如，AAV9的成年啮齿动物，猫，或非人灵长类动物的全身性递送是在脑和脊髓1，2，3个神经元诱导转基因表达仅适度有效。 AAV9载体更有效的鞘内输送也已显示导致在解剖学定义的神经元池中只有有限的转基因表达。更具体地说，它已经是恶魔认为在非人灵长类动物，猪或啮齿动物中的顺式或骶 - 囊性鞘内AAV9传递导致脊髓α运动神经元和节段背根神经节神经元中高水平的转基因表达。然而，在脊柱的interneurons或升序或在白质下行轴突最小的或没有表达看到4个 ，5，6，7。总的来说，这些数据表明，存在高度有效的生物解剖屏障，其防止鞘内递送的AAV扩散进入更深的脊髓实质。 在以前使用成年大鼠和猪的研究中，开发了一种新型的唾液腺载体递送技术8 。使用这种方法，在单次推荐的subpial AAV9递送后证明了高效和多节段的转基因表达。一贯看到强烈的GFP表达在神经元，神经胶质细胞和下降/上升的轴突通过注射的脊髓段。这项研究首次表明，该皮肤表现出限制从鞘内空间扩散到脊髓实质的有效AAV9的主要障碍。虽然这种先前开发的技术和亚型注射装置相对容易在大型啮齿动物（如大鼠）或成年猪中使用，但该系统不适合用于小型动物，例如成年小鼠。由于各种神经变性疾病的可用转基因小鼠模型的数量很多，所以显然需要在小鼠中发展有效的脊髓实质载体递送技术。这种技术的可用性将允许研究特异性基因沉默（ 例如，使用shRNA）或使用细胞非特异性（ 例如巨细胞病毒-CNV或泛素）或细胞特异性（ 例如，突触素或胶质细胞）的上调的作用纤维酸性蛋白质（GFAP））启动子。 因此，在本研究中，我们开发和验证了一种可以有效地用于成年小鼠的微型载体递送系统。类似地，如在以前的大鼠和猪研究中，这项工作在小鼠中单次推注AAV9单次递送后，证明在整个脊髓实质中有力的转基因表达。这种方法的简单性，注射的小鼠对于亚临床AAV9递送的非常好的耐受性以及脊髓实质中转基因表达的高效力表明该技术可以在任何实验室设置中有效地实施并用于靶向脊髓基因表达的实验中。

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subpial adeno-associated virus 9 (aav9) vector delivery in adult mice

已经报道了在成年大鼠和猪中成功开发涎腺腺相关病毒9（AAV9）载体递送技术。已经证明使用低位放置的聚乙烯导管（PE-10或PE-5）用于AAV9递送，通过脊髓实质（白色和灰色物质）在低水平注射的脊髓节段中的有效转基因表达。由于神经变性疾病的转基因小鼠模型的广泛范围，强烈希望在成年小鼠中发展有效的中枢神经系统（CNS） - 靶向载体递送技术。因此，本研究描述了在成年C57BL / 6J小鼠中开发脊髓辅助载体递送装置和技术以允许安全和有效的脊髓AAV9递送。在脊髓固定和麻醉的小鼠中，使用XYZ操纵器用尖锐的34G针切开皮肤（颈部1和腰部1-2脊柱节段水平）。第二个XYZ马然后使用咬合器将平坦的36G针推进到腰部和/或颈部的腋下空间中。然后，首先注射编码绿色荧光蛋白（GFP）的AAV9载体（3-5μL; 1.2×10 13个基因组拷贝（gc））。注射后，定期评估神经功能（运动和感觉），动物在用4％多聚甲醛递送AAV9后14天灌注固定。水平或横向脊髓切片的分析显示在整个脊髓中的转基因表达，包括灰色和白色物质。此外，在运动皮层，细胞核和网状细胞的下降的运动轴突和神经元中观察到强烈的逆行介导的GFP表达。任何动物均未观察到神经功能障碍。这些数据表明，唾液腺向量传递技术可以成功地用于成年小鼠，而不会引起与程序相关的脊髓损伤，并且与高效转基因表达有关整个脊髓神经轴突。

<html> 亚型AAV9注射片段中强有力的转基因表达： 在AAV9递送14天后脊髓切片中转基因（GFP）表达的分析显示整个脊髓实质中具有AAV9-剂量依赖性GFP表达。首先，注射到上腰椎间质空间中的两次双侧3μL注射AAV9-UBI-GFP与全部腰脊髓中白色和灰质的近完全感染相关，延伸至上胸段（ 图2A和2B ，左列和中间列）。两只双侧1.5μL注射...

Watch this Scientific Journal Video about Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice at JoVE.com

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

本研究的目的是通过在成年小鼠中使用新的唾液基因传递技术来开发和验证脊髓腺相关病毒9（AAV9）介导的基因递送的效力和安全性。

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. ...

Research

JoVE Journal

Neuroscience

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

Intracranial Injection of Adeno-associated Viral Vectors

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

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

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

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

腺相关病毒和共聚焦显微镜对器官脑切片胶质细胞器的可视化和实时成像

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

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

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

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), ...

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

在在线 MRI 图像的指导下,对流增强将视遗传阿德诺相关病毒载体输送到 Rhesus Macaque 的皮质

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

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

使用腺相关病毒在小鼠中瞄准科菲林操纵的大脑皮层区域的电皮质记录

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

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

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 ...

DREADDs Technology and Automated Blood Sampling

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Author Spotlight: Automated Infusion and Blood Sampling for Precise Hormonal Analysis in Conscious Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...