Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors
Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors
复制标题
DOI：
10.1113/jphysiol.2010.192971
复制
发表时间：
2010-11-01
影响因子：
5.5
通讯作者：
Undem, B. J.
中科院分区：
文献类型：
--
作者：
Kollarik, M.;Carr, M. J.;Undem, B. J.
支配胸腔结构（如呼吸道和食道）的迷走神经传入纤维是多种多样的，包括几种功能不同的c纤维和a纤维亚型。通过病毒载体对迷走神经传入神经元进行选择性、有效和长期的转导，将进一步推进这些神经亚型的形态学和功能研究。在这里，我们提出了一种假设，即迷走感觉神经元可以用腺相关病毒（AAV）载体在体内转导，这种方式将有助于神经末梢的形态学评估，使用增强的绿色荧光蛋白（eGFP），以及以组织选择性的方式选择性敲除感兴趣的特定基因。我们发现，在体内将AAV载体直接显微注射到迷走神经结节神经节中，可导致绝大多数初级感觉迷走神经神经元的选择性、有效和持久的转导，而没有副交感神经输出神经元的转导。假血清型AAV2/8载体在体内对迷走神经细胞的转导非常有效，因此可以利用短发夹RNA （shRNA）在功能上沉默TRPV1基因的表达。由AAV载体编码的eGFP可被稳健地转运到转导的迷走传入神经元的中枢和外周末梢，从而对活组织中的神经末梢进行清晰的成像，适合于迷走传入神经末梢的结构-功能研究。最后，AAV2/8载体被内脏组织内的迷走神经末梢有效吸收并逆行转运到细胞体，从而实现组织特异性转导。
Vagal afferent fibres innervating thoracic structures such as the respiratory tract and oesophagus are diverse, comprising several subtypes of functionally distinct C-fibres and A-fibres. Both morphological and functional studies of these nerve subtypes would be advanced by selective, effective and long-term transduction of vagal afferent neurons with viral vectors. Here we addressed the hypothesis that vagal sensory neurons can be transduced with adeno-associated virus (AAV) vectors in vivo, in a manner that would be useful for morphological assessment of nerve terminals, using enhanced green fluorescent protein (eGFP), as well as for the selective knock-down of specific genes of interest in a tissue-selective manner. We found that a direct microinjection of AAV vectors into the vagal nodose ganglia in vivo leads to selective, effective and long-lasting transduction of the vast majority of primary sensory vagal neurons without transduction of parasympathetic efferent neurons. The transduction of vagal neurons by pseudoserotype AAV2/8 vectors in vivo is sufficiently efficient such that it can be used to functionally silence TRPV1 gene expression using short hairpin RNA (shRNA). The eGFP encoded by AAV vectors is robustly transported to both the central and peripheral terminals of transduced vagal afferent neurons allowing for bright imaging of the nerve endings in living tissues and suitable for structure-function studies of vagal afferent nerve endings. Finally, the AAV2/8 vectors are efficiently taken up by the vagal nerve terminals in the visceral tissue and retrogradely transported to the cell body, allowing for tissue-specific transduction.