利用工程化锌指蛋白进行光诱导基因调控。
Light-inducible gene regulation with engineered zinc finger proteins.
作者信息
Polstein Lauren R, Gersbach Charles A
机构信息
Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, 90281, Durham, NC, 27708-0281, USA.
出版信息
Methods Mol Biol. 2014;1148:89-107. doi: 10.1007/978-1-4939-0470-9_7.
Abstract
The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells.
摘要
光诱导蛋白质-蛋白质相互作用与基因调控系统的耦合实现了用光来控制基因表达。特别是，来自植物的异源二聚体蛋白质对可用于构建哺乳动物细胞中的基因调控系统，该系统具有可逆性、可重复性、可调性、时空可控性，并且可靶向任何DNA序列。这个系统，即利用工程化锌指蛋白的光诱导转录（LITEZ），基于蓝光诱导的GIGANTEA与FKF1的LOV结构域之间的相互作用，该相互作用驱动转录激活因子定位到高度可定制的工程化锌指蛋白的DNA结合位点。本章提供了修改LITEZ以靶向新DNA序列的方法、设计可编程LED阵列以照射细胞培养物的方法，以及使用修改后的LITEZ系统在哺乳动物细胞中实现转基因表达的时空控制的方法。
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