Journal of Shandong University (Health Sciences) ›› 2026, Vol. 64 ›› Issue (9): 8-16.doi: 10.6040/j.issn.1671-7554.0.2025.1536

• Preclinical Medicine • Previous Articles    

Construction and application of an endogenous LSR-targeted dual-reporter cell line integrating EGFP and Gluc

LIU Hao1, JIAO Peng2, CHEN Liuyan1, GONG Yongfeng1,3, ZHAO Shengtian4, AN Yanan1,3   

  1. 1. Department of Physiology, Shandong Medical and Pharmaceutical University, Yantai 264003, Shandong, China;
    2. Department of Urology, Shandong Medical and Pharmaceutical University, Binzhou 256603, Shandong, China);
    3. Shandong Engineering Research Center of Molecular Medicine for Renal Diseases, Yantai 264003, Shandong, China;
    4. Cutting-Edge Technology Innovation and Transformation Center, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China
  • Published:2026-09-09

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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

CLC Number: 

  • R34
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