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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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远红色光基因编码离子指示器

Rochelin Dalangin1,2, Bill Z Jia3,4, Yitong Qi3

  • 1Department of Chemistry, University of Alberta, Edmonton, AB, Canada.

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概括

研究人员为先进的神经科学研究开发了新的远红色遗传编码离子 (Ca2+) 指标 (GECI). 这些新型GECI能够在体内灵敏地检测神经元活动和细胞过程.

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科学领域:

  • 神经科学是一个神经科学.
  • 分子生物学分子生物学
  • 生物技术是生物技术.

背景情况:

  • 基因编码的离子指标 (GECI) 对于观察单细胞水平的动态和神经元活动至关重要.
  • 现有的GECI通常在光谱范围和灵敏度上有局限性,需要开发新的工具.

研究的目的:

  • 设计和开发新的远红色光GECI,用于增强功能成像.
  • 以亮度,Ca2+亲和力和响应动力学来描述这些新GECI的性能.

主要方法:

  • 开发了两种远红色光GECI,FR-GECO1a和FR-GECO1c,使用单体远红色光蛋白mKelly1和mKelly2.
  • 在实验室中对Ca2+反应的表征,包括动态范围 (ΔF/F) 和明显解离常数 (Kd).
  • 在一光子和两光子照明下评估GECI性能,并评估它们在神经元中检测单动作潜力的能力.

主要成果:

  • FR-GECO1a和FR-GECO1c分别在~596nm和~644nm处表现出激发/发射最大值.
  • 在体外显示显著的Ca2+反应 (ΔF/F0 = 6对于FR-GECO1a,18对于FR-GECO1c) 具有高亲缘关系 (Kd = 29nM对于FR-GECO1a,83nM对于FR-GECO1c).
  • 在单光子和双光子显微镜下,FR-GECO提供明亮信号,并使神经元动作潜力的灵敏,快速检测成为可能.

结论:

  • FR-GECO1a和FR-GECO1c代表了具有提高灵敏度和亮度的先进远红色GECI.
  • 这些工具有助于对神经元活动和细胞过程进行体内成像.
  • 开发的GECI适用于全光学操纵和测量细胞活动,当与光遗传学执行器相结合时.