A highly efficient SFFV promoter-driven mCherry-GFP-LC3B dual-fluorescence system for autophagy monitoring in erythroid cells
10.16352/j.issn.1001-6325.2025.07.0866
- VernacularTitle:SFFV启动子高效驱动mCherry-GFP-LC3B双荧光体系在红系细胞中进行自噬检测
- Author:
Jiuqiang REN
1
;
Jing LI
;
Zhuo LI
;
Xuehui LIU
;
Xiang LYU
Author Information
1. 中国医学科学院基础医学研究所 北京协和医学院基础学院 病理生理学系 疑难重症及罕见病全国重点实验室,北京 100005
- Keywords:
SFFV promoter;
autophagy;
dual-fluorescent mCherry-GFP-LC3B;
erythroid cells
- From:
Basic & Clinical Medicine
2025;45(7):866-873
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a modified lentiviral expression system of mCherry-GFP-LC3B,driven by the hematopoietic-specific SFFV(spleen focus-forming virus)promoter,in order to perform an efficient and real-time monitoring of autophagy flux with in terminally differentiated erythroid cells.Methods The lentiviral plasmid pRSC-SFFV-mCherry-GFP-LC3B was constructed and packaged into lentiviruses for infection of human erythroid progenitor cell line(HUDEP-2)and mouse fetal liver-derived primary erythroid cells.Autophagy dynamics of the cells were then analyzed using fluorescence imaging,Western blot,and flow cytometry in serum starvation and chloroquine inter-vention models.Results The SFFV promoter rendered significantly higher reporter expression efficiency than CMV promoter in HUDEP-2(97%vs.60%)(P＜0.01)and in the primary mouse erythroid cells(83%vs.1%)(P＜0.001),without disrupting normal erythroid differentiation.Serum deprivation increased au-tolysosomes(red puncta),elevated LC3-Ⅱ/LC3-Ⅰratios,and decreased p62 levels.Chloroquine treatment in-duced autophagosome(yellow puncta)accumulation(P＜0.001),showing a dose-dependent inhibition of auto-phagy flux(r2=0.92).Conclusions The SFFV-driven dual-fluorescent system enables robust and real-time mo-nitoring of autophagy flux in erythroid cells,providing a sensitive tool for mechanistic study of erythroid differenti-ation and related disorders such as anemia.
