整合EGFP和Gluc的内源性LSR双报告基因细胞系的构建及应用

doi:10.6040/j.issn.1671-7554.0.2025.1536

摘要/Abstract

摘要： 目的构建一种能同时在转录与翻译水平对脂解刺激脂蛋白受体(lipolysis-stimulated lipoprotein receptor, LSR)进行双重检测的报告系统,以实现对LSR表达和功能的精准监测。方法采用规律间隔成簇短回文重复序列/规律间隔成簇短回文repeat序列相关蛋白系统9(clustered regularly interspaced short pal-indromic repeats associated protein 9, CRISPR/Cas9)介导的基因敲入策略,在HEK293细胞LSR基因最后一个外显子终止密码子上游定点整合增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)和高斯荧光素酶(Gaussia luciferase, Gluc)编码序列,构建双报告基因系统。通过EGFP荧光信号实现LSR表达的实时可视化观察,借助分泌型Gluc的活性检测完成定量分析,并通过内源性LSR敲低结合蛋白质印迹法、免疫荧光染色及实时荧光定量PCR验证系统的可靠性。结果成功建立的LSR-EGFP-Gluc敲入细胞系可准确模拟内源性LSR的表达与功能。EGFP荧光可实时反映LSR的动态表达,Gluc活性检测提供了高灵敏度、高特异性的定量读数。功能验证证实,该细胞系中LSR表达对内源性LSR敲低及蛋白合成抑制剂的应答符合预期规律。通过转录调控因子筛选,鉴定出固醇调节元件结合蛋白2(sterol regulatory element-binding protein 2, SREBP2)和过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ, PPARγ)为LSR表达的转录调控分子。结论本研究构建的LSR双报告基因系统可互补监测LSR动态变化,兼具高灵敏度、准确性与多功能性。

关键词: 脂解刺激脂蛋白受体, CRISPR/Cas9, 增强型绿色荧光蛋白, 高斯荧光素酶, 环己酰亚胺

Abstract: Objective To construct a dual-reporter system capable of detecting lipolysis-stimulated lipoprotein receptor(LSR)expression at both transcriptional and translational levels, thereby achieving accurate monitoring of LSR expression and function. Methods Using a CRISPR/Cas9-mediated gene knock-in strategy, the coding sequences of enhanced green fluorescent protein(EGFP)and Gaussia luciferase(Gluc)were site-specifically integrated upstream of the stop codon in the last exon of the LSR gene in HEK293 cells. Real-time visualization of LSR expression was achieved via EGFP fluorescence, and quantitative analysis was accomplished by measuring the activity of secreted Gluc. The reliability of the system was validated by endogenous LSR knockdown combined with Western blotting, immunofluorescence staining, and quantitative real-time PCR. Results The established LSR-EGFP-Gluc knock-in cell line accurately mimicked the expression and function of endogenous LSR. EGFP fluorescence dynamically reflected LSR expression, while Gluc activity detection provided a highly sensitive and specific quantitative readout. Functional validation confirmed that LSR expression in this cell line responded to endogenous LSR knockdown and protein synthesis inhibition as expected. Screening of transcriptional regulators identified sterol regulatory element-binding protein 2(SREBP2)and peroxisome proliferator-activated receptor γ(PPARγ)as transcriptional regulators of LSR expression. Conclusion The LSR dual-reporter system constructed in this study enables complementary monitoring of dynamic changes in LSR, offering high sensitivity, accuracy, and versatility.

Key words: Lipolysis-stimulated lipoprotein receptor, CRISPR/Cas9, Enhanced green fluorescent protein, Gaussia luciferase, Cycloheximide

中图分类号:

刘浩,矫鹏,陈柳燕,巩永凤,赵升田,安雅男. 整合EGFP和Gluc的内源性LSR双报告基因细胞系的构建及应用[J]. 山东大学学报 (医学版), 2026, 64(9): 8-16.

LIU Hao, JIAO Peng, CHEN Liuyan, GONG Yongfeng, ZHAO Shengtian, AN Yanan. Construction and application of an endogenous LSR-targeted dual-reporter cell line integrating EGFP and Gluc[J]. Journal of Shandong University (Health Sciences), 2026, 64(9): 8-16.

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