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通过光蛋白辅助细胞分类对内源蛋白进行CRISPR-Cas9介导的生物发光标记

Robert G Hawley¹, Teresa S Hawley²

¹Department of Anatomy and Cell Biology, School of Medicine and Health Sciences, George Washington University, Washington, DC, USA. rghawley@gwu.edu.

Methods in molecular biology (Clifton, N.J.)

|March 25, 2024

概括

研究人员开发了一种新方法来寻找针对PAX3-FOXO1融合蛋白的药物,PAX3-FOXO1融合蛋白是膜融合基因阳性狂宫肌肉瘤的关键驱动因素. 这种方法使用发光标签来实现新型癌症治疗的高通量查.

科学领域:

在瘤学瘤学.
分子生物学分子生物学
生物化学生物化学

背景情况:

瘤融合基因是许多癌症的关键驱动因素,呈现出有吸引力的治疗点.
像PAX3-FOXO1这样的转录因子融合在膜融合基因阳性狂宫肌肉瘤 (FP-RMS) 中缺乏明显的药物结合部位,阻碍了向抑制.
目前针对激酶的现有疗法,如对BCR-ABL1的伊马替尼甲基酸,对转录因子融合没有直接适用.

研究的目的:

为了识别选择性准PAX3-FOXO1融合蛋白的新型小分子.
开发一种敏感和定量方法,用于选PAX3-FOXO1抑制剂和降解剂的化合物库.

主要方法:

通过CRISPR-Cas9介导的敲进方法,在FP-RMS细胞系中使用了标记内源PAX3-FOXO1融合蛋白与亲发光HiBiT标记.
一个与mCherry光蛋白相关的P2A自切割被用于基于光激活细胞分类 (FACS) 的单细胞克隆的选择.
HiBiT标签可以对PAX3-FOXO1蛋白水平进行高度敏感的发光检测.

主要成果:

在内源PAX3-FOXO1融合蛋白上成功生成FP-RMS细胞系,具有碳氧终端HiBiT标签.
与传统的光酶相比,HiBiT标签提供了约100倍更明亮的发光信号.
开发的系统允许对复合库进行定量,高通量选.

结论:

对PAX3-FOXO1的HiBiT标记为发现针对FP-RMS的新型治疗剂提供了一个敏感而强大的平台.
这种方法克服了针对转录因子融合的挑战,通过使蛋白质水平的检测成为可能.
该方法方便识别PAX3-FOXO1抑制剂和降解剂,用于潜在的癌症治疗.

关键词:

克里斯普尔-Cas9是什么意思这是一种HiBiT生物发光标签.P2A自切割是P2A的自切割.在PAX3-FOXO1中.光激活细胞分类光激活细胞分类聚变阳性拉布多米索尔科马.m桃光蛋白是一种光蛋白.

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