Watakabe Akiya, Sadakane Osamu, Hata Katsusuke, Ohtsuka Masanari, Takaji Masafumi, Yamamori Tetsuo
Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Dev Neurobiol. 2017 Mar;77(3):354-372. doi: 10.1002/dneu.22459. Epub 2016 Oct 26.
It is important to study the neural connectivities and functions in primates. For this purpose, it is critical to be able to transfer genes to certain neurons in the primate brain so that we can image the neuronal signals and analyze the function of the transferred gene. Toward this end, our team has been developing gene transfer systems using viral vectors. In this review, we summarize our current achievements as follows. 1) We compared the features of gene transfer using five different AAV serotypes in combination with three different promoters, namely, CMV, mouse CaMKII (CaMKII), and human synapsin 1 (hSyn1), in the marmoset cortex with those in the mouse and macaque cortices. 2) We used target-specific double-infection techniques in combination with TET-ON and TET-OFF using lentiviral retrograde vectors for enhanced visualization of neural connections. 3) We used an AAV-mediated gene transfer method to study the transcriptional control for amplifying fluorescent signals using the TET/TRE system in the primate neocortex. We also established systems for shRNA mediated gene targeting in a neocortical region where a gene is significantly expressed and for expressing the gene using the CMV promoter for an unexpressed neocortical area in the primate cortex using AAV vectors to understand the regulation of downstream genes. Our findings have demonstrated the feasibility of using viral vector mediated gene transfer systems for the study of primate cortical circuits using the marmoset as an animal model. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 354-372, 2017.
研究灵长类动物的神经连接性和功能非常重要。为此,能够将基因转移到灵长类动物大脑中的特定神经元至关重要,这样我们就可以对神经元信号进行成像并分析转移基因的功能。为此,我们的团队一直在开发使用病毒载体的基因转移系统。在这篇综述中,我们总结了目前的成果如下。1)我们比较了在绒猴皮层中使用五种不同腺相关病毒血清型与三种不同启动子(即巨细胞病毒(CMV)、小鼠钙/钙调蛋白依赖性蛋白激酶II(CaMKII)和人类突触素1(hSyn1))进行基因转移的特征,以及在小鼠和猕猴皮层中的特征。2)我们使用了靶向特异性双感染技术,并结合使用慢病毒逆行载体的TET-ON和TET-OFF,以增强神经连接的可视化。3)我们使用腺相关病毒介导的基因转移方法,在灵长类新皮层中使用TET/TRE系统研究放大荧光信号的转录控制。我们还建立了在新皮层区域中使用短发夹RNA介导的基因靶向系统,该区域中基因显著表达,并且使用CMV启动子通过腺相关病毒载体在灵长类皮层中未表达的新皮层区域中表达该基因,以了解下游基因的调控。我们的研究结果证明了使用病毒载体介导的基因转移系统,以绒猴作为动物模型来研究灵长类皮层回路的可行性。© 2016威利期刊公司。《发育神经生物学》77: 第354 - 372页,2017年。