颅内注射腺相关病毒载体
Summary
在这里，我们目前颅内注射AAV载体在视觉皮层神经元和神经胶质细胞的荧光标记。
Abstract
颅内的设计，以表达荧光蛋白的病毒载体注射液是一种在体内和不同脑区的脑切片中特定的细胞亚群的可视化的通用标签技术。与荧光染料注射，病毒标签提供针对个别的细胞类型和更便宜和时间消耗比建立转基因小鼠系。在这种技术中，腺相关病毒（AAV）载体注入颅内使用与周围组织的损伤最小立体坐标，一个微管和一个精准投放的AAV的自动泵所需的区域。注射参数，可以根据个人的实验，通过调整注射，注射部位，注射量，注射率，腺相关病毒血清型和基因表达的启动子驾驶的动物年龄。根据选择的条件，可以让病毒诱导的基因表达组细胞，单个细胞或精细胞的过程中，可视化的树突棘水平。这里显示的实验中，描绘了一个双链标签在鼠标的初级视觉皮层的神经元和神经胶质细胞表达绿色荧光蛋白的腺相关病毒注射。
Protocol
1。病毒处理和存储要使用的病毒的生物安全水平的基础上，应选择适当的保护和处理技术。这些做法，可以发现在生物安全微生物和生物医学实验室第 5版，疾病预防控制中心网站上提供 （ http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm ）。 AAV载体的使用已被批准用于1级生物安全（BSL – 1）。实验显示，一个白大褂和手套将处理病?…
Discussion
维拉利介导的基因传递持有神经过程的研究和治疗脑部疾病1,2,3的巨大潜力。这项技术的伟大的通用性，也可以利用荧光标记细胞在体外和体内 4成像。在这里，我们展示了一个使用双链的表达增强型绿色荧光蛋白的腺协会病毒在小鼠的视觉皮层的神经元和神经胶质细胞的转导的详细过程。
虽然这项技术是比较直截了当的，也有一些需要考虑的技…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作成为可能，从美国国立卫生研究院（EY012977），在宝来惠康基金，白厅基金会，和斯隆基金会（AKM）的生物医学科学事业奖助学金。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Stoelting Mouse and Neonatal Rat Adaptor | Stoelting | 51625 | Regular stereotax for securing animals for surgery may be substituted | |
| Extra Fine Bonn Scissors, 8.5cm, straight tip, cutting edge 13mm | Fine Science Tools | 14084-08 | ||
| Eye Dressing Forceps, 10cm, tip width 0.5mm, curved | Fine Science Tools | 11152-10 | ||
| Dumont #5/45 Forceps- Dumoxel Standard Tip, 11cm, angled | Fine Science Tools | 11251-35 | Extra-fine tipped forceps for performing craniotomy | |
| Standard Pattern Forceps, straight, 2.5mmx1.35mmtip, 12cm | Fine Science Tools | 11000-12 | ||
| Microtorque Control Box and Tech2000 handpiece | Ram Products, Inc. | TECH2000ON/OFF | Dental drill | |
| Micro Drill Stainless Steel Burrs 1.4mm tip diameter | Fine Science Tools | 19008-14 | ||
| Wiretrol micropipettes, to deliver 1-5 Ul | VWR International/ Drummond | 5-000-1001 or 53480-287 | ||
| Mineral oil | VWR International | 29447-338 | ||
| Manual Micromanipulator and Tilting Base (right-handed) | World Precision Instruments, Inc. | M3301-M3-R | Used for determining stereotaxic co-ordinates | |
| UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller | World Precision Instruments, Inc. | UMP3-1 | ||
| Sutures | VWR International | 95056-952 | ||
| P-97 Flaming/Brown Micropipette Puller | Sutter Instruments | P-97 | ||
| Tobradex | Available from your institution’s veterinary services |
References
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Tags
Intracranial InjectionAdeno-associated Viral VectorsLabeling TechniqueVisualizationSpecific Subsets Of CellsBrain RegionsIn VivoBrain SectionsFluorescent ProteinViral LabelingIndividual Cell TypesTransgenic Mouse LinesAAV VectorStereotaxic CoordinatesMicropipetteAutomated PumpPrecise DeliveryMinimal DamageInjection ParametersAnimal Age At InjectionInjection LocationVolume Of InjectionRate Of InjectionAAV SerotypePromoter Driving Gene ExpressionVirally-induced Transgene ExpressionVisualization Of Cells And ProcessesDendritic Spines
Citer Cet Article
Lowery, R. L., Majewska, A. K. Intracranial Injection of Adeno-associated Viral Vectors. J. Vis. Exp. (45), e2140, doi:10.3791/2140 (2010).