Genetic mouse models for behavioral analysis through transgenic RNAi technology
Genetic mouse models for behavioral analysis through transgenic RNAi technology
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DOI：
10.1111/j.1601-183x.2008.00412.x
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发表时间：
2008-10-01
影响因子：
2.5
通讯作者：
Kuehn, R.
中科院分区：
文献类型：
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作者：
Delic, S.;Streif, S.;Kuehn, R.
药理学抑制剂和基因敲除小鼠已经发展成为分析特定基因在行为中的作用的常规工具。这两种策略都有局限性，比如仅对一小部分蛋白质可用抑制剂，以及构建特定的小鼠突变体需要大量的努力。最近出现的RNA干扰（RNAi）介导的基因沉默提供了一个快速的选择，可以应用于任何编码基因。我们建立了一种通过将短发夹RNA （short hairpin, sh）载体靶向插入特定基因组位点来高效培养转基因敲低小鼠的方法，并研究了基因沉默在成年小鼠大脑中的效率以及这些小鼠在行为分析中的应用。我们构建了促肾上腺皮质激素释放激素受体1型（Crhr1）、富亮氨酸重复激酶2 （Lrkk2）和嘌呤能受体P2X配体门控离子通道7 （P2rx7）基因的shRNA敲除小鼠，并在幼龄和11月龄敲除小鼠的大脑中发现了shRNA的普遍表达和靶mRNA和蛋白的有效抑制。Crhr1基因敲除小鼠表现出焦虑相关行为减少，应激反应受损，从而概括了Crhr1基因敲除小鼠的表型。我们的研究结果显示了基因沉默在成年大脑中的可行性，并验证了敲低小鼠作为适合行为分析的新遗传模型。
Pharmacological inhibitors and knockout mice have developed into routine tools to analyze the role of specific genes in behavior. Both strategies have limitations like the availability of inhibitors for only a subset of proteins and the large efforts required to construct specific mouse mutants. The recent emergence of RNA interference (RNAi)-mediated gene silencing provides a fast alternative that can be applied to any coding gene. We established an approach for the efficient generation of transgenic knockdown mice by targeted insertion of short hairpin (sh) RNA vectors into a defined genomic locus and studied the efficiency of gene silencing in the adult brain and the utility of such mice for behavioral analysis. We generated shRNA knockdown mice for the corticotropin-releasing hormone receptor type 1 (Crhr1), the leucine-rich repeat kinase 2 (Lrkk2) and the purinergic receptor P2X ligand-gated ion channel 7 (P2rx7) genes and show the ubiquitous expression of shRNA and efficient suppression of the target mRNA and protein in the brain of young and 11-month-old knockdown mice. Knockdown mice for the Crhr1 gene exhibited decreased anxiety-related behavior, an impaired stress response, and thereby recapitulate the phenotype of CRHR1 knockout mice. Our results show the feasibility of gene silencing in the adult brain and validate knockdown mice as new genetic models suitable for behavioral analysis.