Stable Expression of Coagulation Factors by RPS6 Promoter.
10.19746/j.cnki.issn.1009-2137.2023.02.026
- Author:
Wen-Hui ZHANG
1
,
2
,
3
;
Wen-Tian WANG
1
,
2
,
3
;
Ying CHI
1
,
2
,
3
;
Hui-Yuan LI
1
,
2
,
3
;
Feng XUE
1
,
2
,
3
;
Ren-Chi YANG
1
,
2
,
3
;
Lei ZHANG
1
,
2
,
4
Author Information
1. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Hematologic Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College
2. Tianjin Key Laboratory of Gene Therapy for Blood Diseases
3. Key Laboratory of Gene Therapy for Blood Diseases, Chinese Academy of Medical Sciences, Tianjin 300020, China.
4. Key Laboratory of Gene Therapy for Blood Diseases, Chinese Academy of Medical Sciences, Tianjin 300020, China .E-mail: zhanglei1@ihcams.ac.cn.
- Publication Type:Journal Article
- Keywords:
RPS6 promoter;
coagulation factors;
gene therapy;
hemophilia
- MeSH:
Humans;
Transduction, Genetic;
Genetic Vectors;
Hemophilia A/genetics*;
Transfection;
Blood Coagulation Factors/genetics*;
Lentivirus/genetics*
- From:
Journal of Experimental Hematology
2023;31(2):489-494
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia.
METHODS:Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene.
RESULTS:The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups.
CONCLUSION:After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.
