Rapid, Long-Term Labeling of Cells in the Developing and Adult Rodent Visual Cortex Using Double-Stranded Adeno-Associated Viral Vectors
Rapid, Long-Term Labeling of Cells in the Developing and Adult Rodent Visual Cortex Using Double-Stranded Adeno-Associated Viral Vectors
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DOI：
10.1002/dneu.20735
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发表时间：
2009-09-01
影响因子：
3
通讯作者：
Majewska, Ania K.
中科院分区：
文献类型：
--
作者：
Lowery, Rebecca L.;Zhang, Yu;Majewska, Ania K.
脑的慢性体内成像研究需要快速、持久、有效、无毒和细胞类型特异性的标记方法。在过去的十年中，腺相关病毒（AAV）已被用于在体内神经元中稳定表达荧光蛋白。然而，AAV对于许多研究（例如神经元发育的研究）的主要限制是第二链DNA合成的必要性，这延迟了峰值转基因表达。双链AAV（dsAAV）载体的开发已经克服了这一限制，允许快速转基因表达。在这里，我们注射了不同血清型（1，2，6，7，8，和9）的dsAAV载体携带的绿色荧光蛋白（GFP）基因到发育和成年小鼠的视觉皮层和其特征在于其表达。我们观察到标记的神经元和星形胶质细胞的特定的嗜性。dsAAV-GFP标记显示早在注射后2天和长达一个月的神经元GFP表达的高水平，超过了常规AAV的表达起始并匹配其寿命。用dsAAV-GFP标记的神经元在结构和电生理学上与未标记的神经元相同，表明dsAAV-GFP既不具有细胞毒性也不改变正常的神经元功能。我们还证明了dsAAV标记的细胞可以在体内多天内以亚细胞分辨率成像。我们的结论是，双链AAV是一个很好的载体快速标记和长期的星形胶质细胞和神经元在单细胞水平上的体内成像研究的发展和成人的视觉皮层。(C)2009年，Wiley期刊。Inc.开发神经生物学69：674-688，2009
Chronic in vivo imaging studies of the brain require a labeling method that is fast, long-lasting, efficient, nontoxic, and cell-type specific. Over the last decade, adeno-associated virus (AAV) has been used to stably express fluorescent proteins in neurons in vivo. However, AAV's main limitation for many studies (such as those of neuronal development) is the necessity of second-strand DNA synthesis, which delays peak transgene expression. The development of double-stranded AAV (dsAAV) vectors has overcome this limitation, allowing rapid transgene expression. Here, we have injected different serotypes (1, 2, 6, 7, 8, and 9) of a dsAAV vector carrying the green fluorescent protein (GFP) gene into the developing and adult mouse visual cortex and characterized its expression. We observed labeling of both neurons and astrocytes with serotype-specific tropism. dsAAV-GFP labeling showed high levels of neuronal GFP expression as early as 2 days postinjection and as long as a month, surpassing conventional AAV's onset of expression and matching its longevity. Neurons labeled with dsAAV-GFP appeared structurally and electrophysiologically identical to nonlabeled neurons, suggesting that dsAAV-GFP is neither cytotoxic nor alters normal neuronal function. We also demonstrated that dsAAV-labeled cells can be imaged with subcellular resolution in vivo over multiple days. We conclude that dsAAV is an excellent vector for rapid labeling and long-term in vivo imaging studies of astrocytes and neurons on the single cell level within the developing and adult visual cortex. (C) 2009 Wiley Periodicals. Inc. Develop Neurobiol 69: 674-688, 2009