Construction of an ATP probe Chemo-G lentiviral vector and establish-ment of a stable transfection cell line
10.7644/j.issn.1674-9960.2025.04.003
- VernacularTitle:ATP探针Chemo-G慢病毒载体的构建及其稳定转染细胞系的建立
- Author:
Wenjun WU
1
;
Haixin ZHAO
;
Jun GAO
;
Kai WANG
;
Qiuying HAN
;
Teng LI
;
Xin PAN
Author Information
1. 军事科学院军事医学研究院,北京 100850
- Keywords:
adenosine triphosphate(ATP);
cytoplasmic ATP probe;
mitochondrial ATP probe
- From:
Military Medical Sciences
2025;49(4):257-264
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish models for real-time dynamic monitoring of intracellular cytoplasmic adenosine triphosphate(ATP)and mitochondrial ATP levels in cells in order to study the changes in metabolic processes.Methods The lentiviral plasmids of the cytoplasmic chemogenetic green fluorescent protein(GFP)ATP probe(Chemo-G)and those of the mitochondrial-localized chemogenetic GFP ATP probe(mito-Chemo-G)were constructed before being transfected into HEK293T together with the helper plasmids,respectively.Chemo-G and mito-Chemo-G lentiviruses were obtained.HeLa cells were infected with the lentivirus.Puromycin resistance selection and flow cytometry cell sorting were employed to identify and isolate the infected HeLa cells.The growth and GFP expressions of HeLa cells were observed.A live-cell imaging system was used for continuous imaging of the cells,with stimuli added at specific time points to alter intracellular ATP levels to observe changes in the fluorescence intensity of the ATP probe.Results Lentiviral plasmids containing Chemo-G and mito-Chemo-G sequences were constructed.Two cell lines which could stably express Chemo-G and mito-Chemo-G were established that exhibited strong growth and accurate intracellular fluorescence localization.Live-cell imaging revealed that after the addition of 2-deoxy-D-glucose(2-DG)into HeLa-Chemo-G,the fluorescence resonance energy transfer(FRET)/GFP ratio showed a decrease that was partially reversed by the addition of glucose.The FRET/GFP ratio increased after histamine stimulation,but rapidly decreased after the addition of oligomycin.Conclusion The Chemo-G and mito-Chemo-G lentiviral vectors and stably transfected cell lines HeLa-Chemo-G and HeLa-mito-Chemo-G are constructed,which provides reliable experimental models for studying cellular metabolism and changes in intracellular ATP levels.
