Neuro293: rest敲除HEK-293细胞系能够表达神经元限制性基因,用于人类神经生物学和神经元蛋白生物化学的高通量测试。

IF 1.3 Q3 BIOCHEMICAL RESEARCH METHODS
Biology Methods and Protocols Pub Date : 2025-05-10 eCollection Date: 2025-01-01 DOI:10.1093/biomethods/bpaf036
Joshua T Moses, Fahad B Shah, Nicholas M McVay, Dylan E Capes, Christopher C Bosse-Joseph, Jocelyn Salazar, Victoria K Slone, John E Eberth, Jonathan Satin, Andrew N Stewart
{"title":"Neuro293: rest敲除HEK-293细胞系能够表达神经元限制性基因,用于人类神经生物学和神经元蛋白生物化学的高通量测试。","authors":"Joshua T Moses, Fahad B Shah, Nicholas M McVay, Dylan E Capes, Christopher C Bosse-Joseph, Jocelyn Salazar, Victoria K Slone, John E Eberth, Jonathan Satin, Andrew N Stewart","doi":"10.1093/biomethods/bpaf036","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf036"},"PeriodicalIF":1.3000,"publicationDate":"2025-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12202048/pdf/","citationCount":"0","resultStr":"{\"title\":\"Neuro293: A REST-knockout HEK-293 cell line enables the expression of neuron-restricted genes for the high-throughput testing of human neurobiology and the biochemistry of neuronal proteins.\",\"authors\":\"Joshua T Moses, Fahad B Shah, Nicholas M McVay, Dylan E Capes, Christopher C Bosse-Joseph, Jocelyn Salazar, Victoria K Slone, John E Eberth, Jonathan Satin, Andrew N Stewart\",\"doi\":\"10.1093/biomethods/bpaf036\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":36528,\"journal\":{\"name\":\"Biology Methods and Protocols\",\"volume\":\"10 1\",\"pages\":\"bpaf036\"},\"PeriodicalIF\":1.3000,\"publicationDate\":\"2025-05-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12202048/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biology Methods and Protocols\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1093/biomethods/bpaf036\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biology Methods and Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/biomethods/bpaf036","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

摘要

获得成熟神经元仍然是神经生物学有效研究的瓶颈。永生化细胞系仍然需要较长的分化期才能获得神经元样细胞,这可能无法有效分化,并且相对于HEK-293等其他细胞系,质粒转染具有挑战性。为了克服表达成熟神经元蛋白的细胞通路有限的挑战,我们从HEK-293中敲除re1沉默转录因子(REST),创造了一种新的神经元样细胞,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/突触后蛋白、电压门控离子通道、神经元-细胞骨架以及神经递质合成、包装和释放。Western blot证实突触素-1 (Syn1)和Snap-25作为两个神经元限制性蛋白,以及钾通道Kv1.2上调。免疫细胞化学显示,神经丝蛋白200kd在神经293细胞体的单一过程中显著上调和积累。同样,当Syn1在细胞体中增加时,Syn1蛋白在突起末端积累,从Neuro293中挤出。感染腺相关病毒(AAV)后,Neuro293通过Syn1启动子表达报告基因。然而,AAV2质粒的瞬时转染通过不依赖启动子的机制导致泄漏表达。尽管许多电压门控离子通道上调,但Neuro293不具有可兴奋膜。总的来说,在HEK-293中敲除rest诱导了一种快速分裂且易于转染的细胞系,该细胞系表达神经元限制性和成熟的神经元蛋白,可用于高通量生化检测,然而,未经进一步修饰,HEK-293和Neuro293都没有表现出可兴奋膜的特性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Neuro293: A REST-knockout HEK-293 cell line enables the expression of neuron-restricted genes for the high-throughput testing of human neurobiology and the biochemistry of neuronal proteins.

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.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Biology Methods and Protocols
Biology Methods and Protocols Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)
CiteScore
3.80
自引率
2.80%
发文量
28
审稿时长
19 weeks
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信