Department of Biology, and Allen Discovery Center, Tufts University, Medford, MA, USA.
Methods Mol Biol. 2021;2258:93-103. doi: 10.1007/978-1-0716-1174-6_7.
Embryogenesis, as well as regeneration, is increasingly recognized to be orchestrated by an interplay of transcriptional and bioelectric networks. Spatiotemporal patterns of resting potentials direct the size, shape, and locations of numerous organ primordia during patterning. These bioelectrical properties are established by the function of ion channels and pumps that set voltage potentials of individual cells, and gap junctions (electrical synapses) that enable physiological states to propagate across tissue networks. Functional experiments to probe the roles of bioelectrical states can be carried out by targeting endogenous ion channels during development. Here, we describe protocols, optimized for the highly tractable Xenopus laevis embryo, for molecular genetic targeting of ion channels and connexins based on CRISPR, and monitoring of resting potential states using voltage-sensing fluorescent dye. Similar strategies can be adapted to other model species.
胚胎发生以及再生越来越被认为是由转录和生物电网络的相互作用来调控的。静息电位的时空模式在模式形成过程中指导众多器官原基的大小、形状和位置。这些生物电特性是通过离子通道和泵的功能建立的，离子通道和泵可以设定单个细胞的电压势，而间隙连接（电突触）则可以使生理状态在组织网络中传播。通过在发育过程中靶向内源性离子通道进行功能实验，可以探测生物电状态的作用。在这里，我们描述了针对高度可操作的非洲爪蟾（Xenopus laevis）胚胎优化的方案，用于基于 CRISPR 的离子通道和连接蛋白的分子遗传靶向，以及使用电压感应荧光染料监测静息电位状态。类似的策略可以适应其他模式物种。
