Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference
Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference
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DOI：
10.1073/pnas.0406214101
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发表时间：
2004-12-07
影响因子：
11.1
通讯作者：
Cryan, JF
中科院分区：
文献类型：
--
作者：
Thakker, DR;Natt, F;Cryan, JF
基因表达分析表明大脑中越来越多的新基因可以作为治疗神经和精神疾病的潜在靶标。通常，这些基因在大脑中普遍表达，因此可能通过多个脑核的作用导致病理生理状态。目前采用转基因动物体内验证此类靶标的策略非常耗时，并且常常受到发育适应的限制。最近出现了使用病毒介导的 RNA 干扰 (RNAi) 的体细胞基因操作，尽管将目标验证限制在特定的脑核中。我们研究了通过非病毒方式将短干扰 RNA (siRNA) 输注到心室系统中是否能够实现序列特异性基因敲除。通过靶向过度表达 EGFP 的小鼠中的转基因 EGFP，分析了 siRNA 诱导的下调的时间性和程度。观察到 EGFP 的广泛敲低，特别是在邻近或背腹侧和中外侧远离输注部位（背侧第三脑室）的区域，在较远端区域的敲低较小。我们对 RNAi 方法提出了挑战，以产生内源性基因的特异性敲低，该内源性基因编码远离输注部位的区域（腹侧中脑）中的多巴胺转运蛋白（DAT）。成年小鼠中的 DAT-siRNA 输注导致大脑中 DAT mRNA 和蛋白质的显着下调，并且还引发了与输注 GBR-12909（一种药理学选择性 DAT 抑制剂）类似（但延迟）的颞部过度运动反应。这种非病毒 RNAi 方法的应用可能会加速神经精神疾病的靶点验证，这些疾病涉及不同大脑区域的基因的复杂相互作用。
Gene expression analysis implicates an increasing number of novel genes in the brain as potential targets for the treatment of neurological and psychiatric disorders. Frequently, these genes are ubiquitously expressed in the brain and, thus, may contribute to a pathophysiological state through actions in several brain nuclei. Current strategies employing genetically modified animals for in vivo validation of such targets are time-consuming and often limited by developmental adaptations. Somatic gene manipulation using viral-mediated RNA interference (RNAi) has emerged recently, although restricting the target validation to specific brain nuclei. We investigated whether nonviral infusion of short interfering RNA (siRNA) into the ventricular system would enable a sequence-specific gene knockdown. The temporality and extent of siRNA-induced down-regulation were analyzed by targeting a transgene, EGFP, in mice overexpressing EGFP. Extensive knockdown of EGFP was observed, especially in regions adjacent or dorsoventrally and mediolaterally distant to the infusion site (dorsal third ventricle), with lesser knockdown in more distal regions. We challenged our RNAi approach to generate a specific knockdown of an endogenous gene, encoding the dopamine transporter (DAT) in regions (ventral midbrain) far distal to the infusion site. DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein in the brain and also elicited a temporal hyperlocomotor response similar to that (but delayed) obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor. Application of this nonviral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.