Cloning of human ? thalassemic mutation ? IVS II654(C→T) and establishment of its mammalian expression system
- VernacularTitle:人?地中海贫血IVSⅡ654(C→T)突变基因的克隆和真核表达体系的构建
- Author:
Shan DUAN
;
Xiaowu FANG
;
Luming CHEN
;
Ruiping ZENG
;
Chuanshu DU
- Publication Type:Journal Article
- Keywords:
Thalassemia;
?IVS II654 mutation gene;
Gene therapy;
Leukemia, erythroblastic, acute
- From:
Chinese Journal of Pathophysiology
1986;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To clone human ?-globin gene carrying a thalassemic mutation IVS II654(C→T) and establish a eukaryotic expression system for high-level expression of human ? IVS II654 gene in mouse erythroleukaemia(MEL) cells. METHODS: The fragments of human ? 654 gene isolated from the ? thalassemia patients homozygous for the ? 654 mutation were amplified by PCR, and cloned to plasmid pBGT51. Then, the human ? LCR and ? 654 gene were subcloned from plasmid pBGT51 to the stable mammalian expression vector pcDNA3.1+ together, and the MEL cells were transfected with this vector using commercially available cationic lipid FuGENE6. The MEL cells were induced for further maturation by DMSO and the expression of human ? 654 gene in the MEL cells was identified by RT-PCR. RESULTS: A mammalian expression system of human ? thalassemic mutation ?IVS II654(C→T) was established. CONCLUSION: The level and the reliability of expression of human ? 654 gene in the MEL cells in vitro are similar to that in vivo in human body. This may be a valuable gene therapy model for human ? thalassemic mutation ?IVS II654(C→T).
