Moses Joshua T, Shah Fahad B, McVay Nicholas M, Capes Dylan E, Bosse-Joseph Christopher C, Salazar Jocelyn, Slone Victoria K, Eberth John E, Satin Jonathan, Stewart Andrew N
Department of Physiology, University of Kentucky, Lexington, KY 40536, United States.
Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY 40536, United States.
Biol Methods Protoc. 2025 May 10;10(1):bpaf036. doi: 10.1093/biomethods/bpaf036. eCollection 2025.
Efficient interrogation of neurobiology remains bottlenecked by obtaining mature neurons. Immortalized cell lines still require lengthy differentiation periods to obtain neuron-like cells, which may not efficiently differentiate and are challenging to transfect with plasmids relative to other cell lines such as HEK-293's. To overcome challenges with limited access to cells that express mature neuronal proteins, we knocked out the RE1-silencing transcription factor (REST) from HEK-293's to create a novel neuron-like cell, which we name Neuro293. RNA-sequencing and bioinformatics analyses revealed a significant upregulation of genes associated with neurobiology and membrane excitability including pre-/post-synaptic proteins, voltage gated ion channels, neuron-cytoskeleton, as well as neurotransmitter synthesis, packaging, and release. Western blot validated the upregulation of Synapsin-1 (Syn1) and Snap-25 as two neuron-restricted proteins, as well as the potassium channel Kv1.2. Immunocytochemistry against Neurofilament 200 kd revealed a significant upregulation and accumulation in singular processes extending from Neuro293's cell body. Similarly, while Syn1 increased in the cell body, Syn1 protein accumulated at the ends of processes extruding from Neuro293's. Neuro293's express reporter-genes through the Syn1 promoter after infection with adeno-associated viruses (AAV). However, transient transfection with AAV2 plasmids led to leaky expression through promoter-independent mechanisms. Despite an upregulation of many voltage-gated ion channels, Neuro293's do not possess excitable membranes. Collectively, REST-knockout in HEK-293's induces a quickly dividing and easily transfectable cell line that expresses neuron-restricted and mature neuronal proteins which can be used for high-throughput biochemical interrogation, however, without further modifications neither HEK-293's or Neuro293's exhibit properties of excitable membranes.
获取成熟神经元仍然是有效研究神经生物学的瓶颈。永生化细胞系仍需要较长的分化时间来获得类神经元细胞，这些细胞可能无法有效分化，并且相对于其他细胞系（如HEK - 293细胞系）而言，用质粒转染具有挑战性。为了克服获取表达成熟神经元蛋白的细胞受限的挑战，我们从HEK - 293细胞中敲除了RE1沉默转录因子（REST），以创建一种新型的类神经元细胞，我们将其命名为Neuro293。RNA测序和生物信息学分析显示，与神经生物学和膜兴奋性相关的基因显著上调，包括突触前/后蛋白、电压门控离子通道、神经元细胞骨架以及神经递质的合成、包装和释放。蛋白质免疫印迹法验证了突触素-1（Syn1）和突触结合蛋白-25（Snap - 25）这两种神经元特异性蛋白以及钾通道Kv1.2的上调。针对200kd神经丝的免疫细胞化学显示，从Neuro293细胞体延伸出的单个突起中显著上调并积累。同样，虽然Syn1在细胞体中增加，但Syn1蛋白在从Neuro293伸出的突起末端积累。在用腺相关病毒（AAV）感染后，Neuro293通过Syn1启动子表达报告基因。然而，用AAV2质粒进行瞬时转染会通过启动子非依赖机制导致渗漏表达。尽管许多电压门控离子通道上调，但Neuro293不具有可兴奋膜。总体而言，HEK - 293细胞中的REST基因敲除诱导出一种快速分裂且易于转染的细胞系，该细胞系表达神经元特异性和成熟的神经元蛋白，可用于高通量生化研究，然而，未经进一步修饰的HEK - 293细胞或Neuro293细胞均不表现出可兴奋膜的特性。
