A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells

doi:10.1186/s12964-019-0359-x

	A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells
	Li, Fei1,3 ; Jiang, Xi 3; Luo, Ling-Ling1 ; Xu, Yue-Ming2 ; Huang, Xing-Xu1 ; Huang, Cheng 3; Zhang, Yu1
	2019-05-23
发表期刊	CELL COMMUNICATION AND SIGNALING
ISSN	1478-811X
卷号	17
发表状态	已发表
DOI	10.1186/s12964-019-0359-x
摘要	BackgroundGPCRs are considered essential for various physiological processes and have been the most productive drug targets. Therefore, development of the methods of GPCR ligands screening is a high priority for pharmaceutical industries and research institutions.MethodsWe developed a potential method (piggyBac-TANGO) based on the TANGO and PRESTO-TANGO assays. The system was optimized with a piggyBac transposon as a transgene vehicle, and eGFP was used as a reporter instead of luciferase. The assay was validated in the HEK 293T and U87-MG cell lines and antagonist activities of the compounds were assessed. The transgene copy number and long-term stability were evaluated by qPCR. Then, we performed a DRD2-targeted screening for natural products using the piggyBac-TANGO assay.ResultsThe validation assay showed that using the piggyBac transposon as a transgene vehicle produced high signal-to-background ratio and stable readout confirmed by investigation of the transgene copy number and long-term stability. Use of eGFP instead of luciferase as a reporter enabled to create a high throughput system suitable for live cells. Moreover, the piggyBac-TANGO assay permitted versatile detection of antagonist activity of compounds and was not limited to a particular cell type. With the use of the piggyBac-TANGO assay, we have successfully identified a novel agonist of DRD2.ConclusionThus, the results indicate that the piggyBac-TANGO method is a user-friendly, robust and imaging-based assay that provides a novel approach to high throughput GPCR-targeted ligand screening and drug development.
关键词	GPCRs Ligand piggyBac-TANGO High signal-to-background ratio Stable readout Live cells
收录类别	SCI ; SCIE
语种	英语
资助项目	Shanghai science and technology development foundation (Shanghai Sailing Program)[17YF1412300]
WOS研究方向	Cell Biology
WOS类目	Cell Biology
WOS记录号	WOS:000468935600001
出版者	BMC
WOS关键词	PROTEIN-COUPLED-RECEPTORS ; GENE-TRANSFER ; TRANSPOSON ; INTERROGATION ; TARGETS ; TOOLS
原始文献类型	Article
引用统计
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/45637
专题	iHuman研究所_公共科研平台_IT平台生命科学与技术学院_PI研究组_黄行许组
通讯作者	Huang, Cheng; Zhang, Yu
作者单位	1.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China 2.ShanghaiTech Univ, iHuman Inst, Shanghai, Peoples R China 3.Shanghai Univ Tradit Chinese Med, Sch Pharm, Shanghai, Peoples R China
第一作者单位	生命科学与技术学院
通讯作者单位	生命科学与技术学院
第一作者的第一单位	生命科学与技术学院
推荐引用方式 GB/T 7714	Li, Fei,Jiang, Xi,Luo, Ling-Ling,et al. A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells[J]. CELL COMMUNICATION AND SIGNALING,2019,17.
APA	Li, Fei.,Jiang, Xi.,Luo, Ling-Ling.,Xu, Yue-Ming.,Huang, Xing-Xu.,...&Zhang, Yu.(2019).A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells.CELL COMMUNICATION AND SIGNALING,17.
MLA	Li, Fei,et al."A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells".CELL COMMUNICATION AND SIGNALING 17(2019).