Generation of a novel HEK293 luciferase reporter cell line by CRISPR/Cas9-mediated site-specific integration in the genome to explore the transcriptional regulation of the PGRN gene

Progranulin has multiple functions in several physiological and pathological processes, including embryonic development, wound repair, tumorigenesis, inflammation and neurodegeneration. To investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK293 cells by integrating luciferase gene in the genome controlled by the endogenous PGRN promoter using CRISPR/Cas9. PCR results demonstrated the site-specific integration of the exogenous luciferase gene into the genome. To validate the novel luciferase knock-in system, a CRISPR/Cas9 transcription activation/repression system for the PGRN gene was constructed and applied to the knock-in system. In addition, phorbol ester (phorbol 12-myristate, 13-acetate), previously reported as activating the expression of PGRN, was applied to the system. The results indicated that luciferase activity was directly correlated with the activity of the PGRN endogenous promoter. This novel system will be a useful tool for investigating the transcriptional regulation of PGRN, and it has great potential in screening the drugs targeting PGRN.

促生长素在多种生理和病理过程中扮演着多重角色，包括胚胎发育、伤口修复、肿瘤发生、炎症和神经退行性变。为探究PGRN基因的转录调控机制，本研究在HEK293细胞中建立了基于CRISPR/Cas9技术的荧光素酶敲入报告系统，通过整合受内源性PGRN启动子调控的荧光素酶基因至基因组中。PCR检测结果证实了外源荧光素酶基因在基因组中的特异性整合。为进一步验证该新型荧光素酶敲入系统，构建了针对PGRN基因的CRISPR/Cas9转录激活/抑制系统，并将其应用于敲入系统中。此外，应用了先前报道能激活PGRN表达的佛波酯（佛波-12-肉豆蔻酸，13-乙酸酯）。实验结果显示，荧光素酶活性与PGRN内源性启动子的活性呈直接相关性。该新型系统将成为研究PGRN转录调控的有用工具，并在筛选针对PGRN的药物方面具有巨大的应用潜力。