NanoDam Profiling of EGL-43 and FOS-1 in Dopaminergic Neurons

Activity-dependent mechanisms have been long known to play a critical role in synaptogenesis. Here, we utilize a recently adapted technology, NanoDam, to perform targeted DamID using endogenously tagged GFP transcription factors. Overall design: A single copy insertion of a dopaminergic promoter driving expression of a GFP nanobody fused to Dam methylase was crossed together with animals carrying endogenous EGL-43::GFP and FOS-1::GFP. Dam methylase permanently methylated DNA in close proximity to regions of TF binding. DNA from L3 worms was extracted, digested with DpnI, amplified, and sequenced using the MinION Nanopore platform

活动依赖性机制在突触发生（synaptogenesis）过程中的关键作用早已被学界公认。本研究借助新近优化的NanoDam技术，利用内源性标记绿色荧光蛋白（GFP）的转录因子开展靶向DamID实验。实验整体设计：将携带多巴胺能启动子驱动与Dam甲基化酶融合的GFP纳米抗体表达的单拷贝插入片段，与携带内源性EGL-43::GFP及FOS-1::GFP的线虫品系进行杂交。Dam甲基化酶可在转录因子（Transcription Factor, TF）结合区域的邻近位置对DNA实施永久性甲基化修饰。研究人员提取三龄线虫（L3）的基因组DNA，经DpnI限制性内切酶消化、扩增后，利用MinION纳米孔测序平台完成测序。