新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导
Summary
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.
Abstract
随着科技进步的步伐迅速加快，需要对实验神经科学的新方法，能够快速，轻松地操纵基因表达的小鼠大脑。在这里，我们描述了一种技术，最早由Passini和沃尔夫介绍了颅内直接供货病毒编码的转基因到新生小鼠大脑。在其最基本的形式中，该过程仅需要一个冰桶和微升注射器。然而，该协议也可适于与立体定位框架上使用，以提高用于研究新的技术一致。该方法依赖于重组腺相关病毒（AAV），以从脑室自由移动进入脑实质，而室管膜衬里仍然是在出生后的第12-24小时期间，未成熟的能力。脑室注射的AAV在这个年龄导致的神经元在整个大脑的普遍转导。在注射几天表达开始，持续了lifetimE中的动物。病毒滴度可以调节，以控制转导神经元的密度，而共表达的荧光蛋白的规定转导的细胞的一个重要的标签。随着病毒核心设施的上升可用性，以提供现成的货架，预包装试剂和自定义病毒制剂，这种方法提供了及时的方法在小鼠大脑中快速，方便，便宜得多操纵基因表达比传统的生殖工程。
Introduction
传统的方法用于修改神经基因表达需要耗时和昂贵的种系操作。另一种从头方法，如在子宫内电穿孔或立体定位慢病毒注射液产生更快的结果，而且成本较低，但有需要复杂的外科手术1-3的劣势。此外，转基因表达具有有限的空间范围使用这些方法。在此，我们通过脑室注射腺相关病毒（AAV）到新生小鼠大脑描述了一种快速，简便，经济的方法进行广泛的神经元处理。该方法最初是由约翰·沃尔夫和Marco Passini于2001年，在那里他们建议的小粒径的AAV允许它在脑脊髓液中的扩散，因为它从侧脑室穿过未成熟的室管膜屏障并进入脑实质4描述的， 5。脑室注射腺相关病毒的F内IRST的出生国债收益率神经亚群跨越大脑的各个区域，从嗅球脑干6,7普遍病毒转导后24小时。病毒交付的转基因表达，并在注射几天活跃，持续长达一年后转。因此，这种多功能的操作使研究从出生后早期大脑发育到衰老和退变的成人。
在适应的技术，我们的具体实验需要，我们主要侧重于AAV8血清型，因为它是最有效的传感神经元6。我们表明，病毒滴度可以稀释，以控制转导神经细胞的遗传操作的实验检测细胞内在后果的密度。此外，我们证明，这两种病毒可以被共注射，以产生被偏向不同的或重叠的集合的神经元的表达模式，这取决于所选择的血清型病毒包装。我们的工作扩展这种技术在范围广泛的实验神经科学设置使用的通用性。
Protocol
Representative Results
Discussion
我们已经描述了一种通用的程序，用于操纵使用AAV作为广泛递送到新生小鼠脑部的车辆神经元基因表达。与神经元的转基因的其它方法，如在子宫内电1或立体定向颅内注射2,3相比，新生病毒注射是比较容易和简单。基本过程可以在几分钟内完成，只有一个冰桶和微升注射器。最佳的生存和转基因表达可以通过参加几个技术细节来实现，最重要的是病毒性的股票，时机和注射的准?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by the Robert A. and Rene E. Belfer Family Foundation, NIA R21 AG038856 (JLJ), BrightFocus Foundation Alzheimer’s Disease research grant A2010097 (JLJ), and NIA Biology of Aging Training grant T32 AG000183 (support for SDG).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| ICR outbred mice | Harlan | Hsd:ICR (CD-1) | This strain is also known as CD-1 |
| FVB inbred mice | The Jackson Laboratory | 1800 | 5-6 weeks of age |
| Nestlets | Lab Supply | NESTLETS | |
| Shepherd shacks | Lab Supply | SS-mouse | |
| High fat rodent chow | Purina Mills | PicoLab Mouse diet 20, #5058 | This is our standard breeder chow |
| High fat rodent chow (alternative) | Harlan Laboratories | Teklad Global 19% protein rodent diet #2019S | If low phytoestrogen, autoclavable diet is needed |
| Injection syringe | Hamilton | 7653-01 | 10 ml syringe |
| Injection needles | Hamilton | 7803-04, RN 6PK PT4 | 32 gauge, for standard P0 injections |
| Metal plate for cryoanesthesia | McMaster Carr | 8975K439 | Raw aluminum plate, 6” x 12”, 0.25” thick, will need to be cut into 3 equal pieces and edges sanded by local machine shop |
| Small animal stereotaxic device with digital readout | David Kopf Instruments | Model 940 | |
| Universal syringe holder with needle support foot | David Kopf Instruments | Model 1772-F1 | |
| Neonatal frame | Stoelting | 51625 | Officially called a mouse and neonatal rat adaptor |
| Biohazard disposal bags with sterile indicator | VWR | 14220-030 | Important! – Check with local veterinary and environmental safety staff to learn your institute’s protocol for biohazard waste disposal |
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