Ren Chonghua, Adams Alexa N, Pyles Benjamin, Bailus Barbara J, O'Geen Henriette, Segal David J
Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, USA.
Guangzhou Key Laboratory of Insect Development Regulation and Application Research, South China Normal University, Guangzhou, China.
Methods Mol Biol. 2018;1867:239-251. doi: 10.1007/978-1-4939-8799-3_18.
Artificial transcription factors based on zinc finger, TALE, and CRISPR/Cas9 programmable DNA-binding platforms have been widely used to regulate the expression of specific genes in cultured cells, but their delivery into organs such as the brain represents a critical challenge to apply such tools in live animals. In previous work, we developed a zinc-finger-based artificial transcription factor harboring a cell-penetrating peptide (CPP) that could be injected systemically, cross the blood-brain barrier, and alter expression of a specific gene in the brain of an adult mouse. Importantly, our mode of delivery produced widespread distribution throughout the brain. Here we describe methods for the production and purification of the factor, testing CPP activity in cells, and testing CPP activity in mice.
基于锌指、转录激活样效应因子（TALE）和CRISPR/Cas9可编程DNA结合平台的人工转录因子已被广泛用于调控培养细胞中特定基因的表达，但将它们递送至大脑等器官是在活体动物中应用此类工具的一项关键挑战。在之前的工作中，我们开发了一种基于锌指的人工转录因子，其含有一种细胞穿透肽（CPP），该因子可通过全身注射，穿过血脑屏障，并改变成年小鼠大脑中特定基因的表达。重要的是，我们的递送方式在整个大脑中产生了广泛分布。在此，我们描述该因子的生产和纯化方法、在细胞中测试CPP活性以及在小鼠中测试CPP活性的方法。
