RNAi-induced gene silencing by local electroporation in targeting brain region
RNAi-induced gene silencing by local electroporation in targeting brain region
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DOI：
10.1152/jn.00161.2004
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发表时间：
2005-01-01
影响因子：
2.5
通讯作者：
Tsumoto, T
中科院分区：
文献类型：
--
作者：
Akaneya, Y;Jiang, B;Tsumoto, T
用于“敲除”（KO）的遗传操作是用于表征靶基因的有用工具。然而，需要克服的缺点阻碍了它在普通实验室中的简单易用。在这里，我们描述了一种敲除技术称为RNA干扰（RNAi）诱导的基因沉默通过局部电穿孔（RISLE）。通过电穿孔将小干扰RNA（siRNA）导入特定脑区域，导致靶基因如GluR 2和考克斯-1的mRNA和蛋白质的表达水平显著降低，而不影响靶蛋白质以外的蛋白质的表达水平或在靶组织中引起病理变化。有效的电脉冲相对较弱，由串联施加的强短脉冲和弱长脉冲组成。RISLE可以敲低目标区域的基因，例如视觉皮层和海马体的CA 1区域，而不会影响其他区域。此外，使用这种技术构建的敲除模型具有与先前发现一致的生理功能，即从突触前位点释放谷氨酸、长时程增强（LTP）和长时程抑制（LTD）。这些结果表明，这种技术是适用的，其特点是空间的灵活性，时间的可访问性，并易于建立击倒模型。组织的完整性是由于施加的微弱电脉冲和有限的基因沉默区域。因此，RISLE可能适用于未来的疾病治疗。
Genetic manipulation for "knockout" (KO) is a useful tool for characterizing a target gene. However, its shortcomings that need to be overcome hinder its easy and ready usage in ordinary laboratories. Here we describe a knockdown technique termed the RNA interference (RNAi)-induced gene silencing by local electroporation (RISLE). Small interfering RNA (siRNA) introduction by electroporation into a specific brain region results in a marked reduction in the expression levels of both the mRNA and protein of the target genes such as GluR2 and Cox-1 without affecting the expression levels of proteins other than that of the target protein or causing pathological changes in the target tissues. The effective electrical pulses are relatively weak, consisting of a strong short pulse and a weak long pulse applied in tandem. RISLE can knock down a gene at the target region, for example, the visual cortex and the CA1 region of the hippocampus, without affecting other regions. Moreover, the knockdown models constructed using this technique have physiological functions consistent with previous findings, that is, glutamate release from presynaptic sites, long-term potentiation (LTP), and long-term depression (LTD). These results suggest that this technique is applicable and characterized by spatial flexibility, temporal accessibility, and ease of establishment of knockdown models. The intactness of the tissue subjected to RISLE is due to the weak electrical pulses applied and the limited area of gene silencing. Thus RISLE may be applicable to disease therapy in the future.