Using genome editing of induced pluripotent stem cells to investigate the genetics of neuronal network function.

神经分化和神经网络的形成是一个精细平衡的过程，对认知功能至关重要，它依赖于基因表达的严格调控。本项目将结合crispr /Cas9强大的基因组编辑新工具和人类诱导多能干细胞（iPS）向神经元分化的能力，研究基因表达在神经元分化中的作用。在控制转录、RNA加工和蛋白质合成的基因中，与脑癌和神经发育障碍有因果关系的突变证明了神经系统中基因表达调节的重要性。基因表达调控因子突变引起的缺陷通常是复杂的，反映了调控因子控制的靶基因的作用。我们最近发现了蛋白质合成的关键调控因子和参与突触活动和神经元细胞骨架形成的靶基因，这些过程对神经元网络的正常功能至关重要[1-3]。该项目将研究这些基因在神经元分化中的作用。这些基因将在人类iPS细胞中使用Crispr/Cas9技术被破坏。iPS细胞可以分化为多能神经祖细胞，并进一步分化为能够形成网络的特定神经元类型。通过比较除Crispr/Cas9诱导突变外完全相同的等基因细胞系衍生的神经元，这种方法可以确定单个基因的作用。网络形成之后，将通过高分辨率显微镜、定量PCR基因表达分析、Western blotting和核糖体分析以及神经元电生理特性分析来分析神经元结构和特性的发展。这些强大的新工具的应用将为神经网络形成的遗传学提供基本的见解。成功的候选人将接受iPS细胞培养和分化、使用Crispr/Cas9技术进行基因组编辑、使用基于定量PCR的方法进行基因表达分析、核糖体分析或Western blotting、先进的显微镜技术和电生理学等方面的培训。培训将由阿伯丁和爱丁堡的参与主管酌情提供。

Neural differentiation and the formation of neuronal networks is a finely balanced process essential for cognitive function that relies on stringent regulation of gene expression. This project will combine the powerful new tool of genome editing by Cripsr/Cas9 with the ability of human induced pluripotent stem (iPS) cells to differentiate to neurons to investigate the role of gene expression in neuronal differentiation. Mutations causally linked to brain cancer and to neurodevelopmental disorders in genes that control transcription, RNA processing and protein synthesis, demonstrate the importance of the regulation of gene expression in the nervous system. Defects caused by mutation of gene expression regulators are normally complex and reflect the roles of the target genes controlled by the regulators. We have recently identified critical regulators of protein synthesis and target genes which are involved in synapse activity and the formation of the neuronal cytoskeleton, processes essential for the normal function of neuronal networks [1-3]. The project will examine the role of these genes in neuronal differentiation. The genes will be disrupted in human iPS cells using Crispr/Cas9 technology. iPS cells can be differentiated to pluripotent neural progenitor cells and further to specific neuron types able to form networks. By comparing neurons derived from isogenic cell lines that are identical except for the Crispr/Cas9 -induced mutation, this approach allows to identify the role of single genes. Network formation will be followed by analysing the development of neuronal structures and properties by a combination of high resolution microscopy, gene expression analysis by quantitative PCR, Western blotting and ribosome profiling, and the analysis of electrophysiological properties of the neurons. The application of these powerful new tools will provide fundamental insight into the genetics of neuronal network formation.The successful candidate will be trained in iPS cell culture and differentiation, genome editing using Crispr/Cas9 technology, the analysis of gene expression using quantitative PCR based approaches, ribosome profiling or Western blotting, and advanced microscopy techniques, and electrophysiology. Training will be provided by the participating supervisors in Aberdeen and Edinburgh as appropriate.