Department of Systems Pharmacology and Translational Therapeutics, Institute for Translational Medicine and Therapeutics, Philadelphia, PA, USA.
Department of Anatomy & Neurobiology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
Methods Mol Biol. 2024;2842:309-321. doi: 10.1007/978-1-0716-4051-7_16.
Modern neuroscience research is increasingly discovering that alterations in epigenetic states within key brain cells is correlated with brain diseases. These epigenetic alterations may include changes in histone post-translational modifications and/or DNA modifications, all of which affect transcription and other gene expression programs within the brain cells that comprise central brain regions. However, the exact causal contribution of these epigenome changes to brain disease cannot be elucidated in the absence of direct in vivo manipulations in the implicated brain areas. Combining the design and creation of epigenetic editing constructs, gene delivery strategies, and stereotaxic surgery enables neuroscience researchers to target and manipulate the epigenetic state of the brain cells of laboratory rodents in a locus-specific manner and test its causal contribution to disease-related pathology and behaviors. Here, we describe the surgical protocol utilized by our group and others, which is optimized for herpes simplex virus delivery into the mouse brain, although the protocol outlined herein could be applied for delivery of adeno-associated viruses, lentiviruses, or nonviral gene-delivery methods in both mice and rats. The method allows for the overexpression of engineered DNA-binding proteins for direct and targeted epigenome editing in rodent brain with excellent spatiotemporal control. Nearly any brain region of interest can be targeted in rodents at every stage of postnatal life. Owing to the versatility, reproducibility, and utility of this technique, it is an important method for any laboratory interested in studying the cellular, circuit, and behavioral consequences of manipulating the brain epigenome in laboratory rodents.
现代神经科学研究越来越发现，关键脑细胞中的表观遗传状态的改变与脑部疾病有关。这些表观遗传改变可能包括组蛋白翻译后修饰和/或 DNA 修饰的变化，所有这些都会影响组成中枢神经系统的脑细胞中的转录和其他基因表达程序。然而，如果不能在受影响的脑区进行直接的体内操作，就无法阐明这些表观基因组变化对脑部疾病的确切因果贡献。结合设计和创建表观遗传编辑构建体、基因传递策略和立体定向手术，使神经科学研究人员能够以特定位置的方式靶向和操纵实验动物脑细胞的表观遗传状态，并测试其对与疾病相关的病理学和行为的因果贡献。在这里，我们描述了我们小组和其他小组使用的手术方案，该方案针对单纯疱疹病毒（HSV）递送至小鼠大脑进行了优化，尽管本文中概述的方案可应用于腺相关病毒（AAV）、慢病毒或非病毒基因传递方法在小鼠和大鼠中的传递。该方法允许过表达工程 DNA 结合蛋白，以在啮齿动物大脑中进行直接和靶向的表观基因组编辑，具有出色的时空控制。在出生后的各个阶段，几乎可以靶向啮齿动物的任何感兴趣的脑区。由于该技术的多功能性、可重复性和实用性，它是任何对研究在实验动物大脑中操纵表观基因组对细胞、回路和行为的影响感兴趣的实验室的重要方法。
