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
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?...

<ol>
	<li>Foust, K., Nurre, E., Montgomery, C., Hernandez, A., Chan, C., Kaspar, B. <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>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. Ther. 18 (12), 2064-2074 (2010).</li><li>Dayton, R., 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=Selective+forelimb+impairment+in+rats+expressing+a+pathological+TDP-43+25+kDa+C-terminal+fragment+to+mimic+amyotrophic+lateral+sclerosis.">Selective forelimb impairment in rats expressing a pathological TDP-43 25 kDa C-terminal fragment to mimic amyotrophic lateral sclerosis.</a> Mol. Ther. 21 (7), 1324-1334 (2013).</li><li>Jackson, K., Dayton, R., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV9+supports+wide-scale+transduction+of+the+CNS+and+TDP-43+disease+modeling+in+adult+rats.">AAV9 supports wide-scale transduction of the CNS and TDP-43 disease modeling in adult rats.</a> Mol. Ther. Methods Clin. Dev. 2, 15036 (2015).</li><li>Jackson, 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=Preservation+of+forelimb+function+by+UPF1+gene+therapy+in+a+rat+model+of+TDP-43-induced+paralysis.">Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced paralysis.</a> Gene Therapy. 22 (1), 20-28 (2015).</li><li>Jackson, K., Dayton, R., Deverman, B., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Better+targeting,+better+efficiency+for+wide-scale+neuronal+transduction+with+the+synapsin+promoter+and+AAV-PHP.B.">Better targeting, better efficiency for wide-scale neuronal transduction with the synapsin promoter and AAV-PHP.B.</a> Front. Mol. Neurosci. 9, 116 (2016).</li><li>Jackson, 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=Severe+respiratory+changes+at+end+stage+in+a+FUS-induced+disease+state+in+adult+rats.">Severe respiratory changes at end stage in a FUS-induced disease state in adult rats.</a> BMC Neuroscience. 17 (1), 69 (2016).</li><li>Xu, L., 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=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>Jackson, K., Dayton, R., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+vector+&amp;#34;magic+bullet&amp;#34;:+targeted+expression+in+the+central+nervous+system+after+peripheral+delivery+using+the+synapsin+promoter.">Gene vector &amp;#34;magic bullet&amp;#34;: targeted expression in the central nervous system after peripheral delivery using the synapsin promoter.</a> Expert Opin Ther Targets. 20 (10), 1153-1154 (2016).</li><li>van Hulst, R., Klein, J., Lachmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+embolism:+pathophysiology+and+treatment.">Gas embolism: pathophysiology and treatment.</a> Clin. Physiol. Funct. Imaging. 23 (5), 237-246 (2003).</li></ol>

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), ...

Research

JoVE Journal

Neuroscience

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

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

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

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

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

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 Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

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

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

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

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

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery 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. ...