Cloning Of Human Thalassemic Mutation β～(41/42) And Establishment Of Its Mammalian Expression Systerm

Background: The thalassemias are a heterogeneous group of inherited disorders.The main pathological mechanism which causes thalassemias is the imbalance in the synthesis of a and β globin chains, due to mutations in the globin gene loci.pthalassemia is the most common hemoglobin disorder that affects population among the southeast Asia including southwest China.The large β^41/42mutation gene is one of the most common genetic defects causing severe pthalassemia in these area.Objective: To clone human β-globin gene carrying a thalassemic mutationβ^41/42 and establish a eukaryotic expression system for high-level expression of human β^41/42gene in mouse erythrodeukaemia (MEL) cells.Methods: The fragments of LCR and the human thalassemic β^41/42 mutation Gene isolated from the beta thalassemia patients homozygous for the β^41/42 mutation were amplified by PCR, and cloned into the stable mammalian expression vector pcDNA3.1(-) together, The recombinant plasmid was certified by enzyme-digestion and sequencing.Then the MEL cells were transfected with this vector using Lipofectamine 2000.The MEL cells were induced for further maturation by DMSO and the expression system of human β^41/42 gene was identified by RT-PCR.Results:We successfully constructed the recombinant plasmid pcDNA3.1-LCR-β^41/42, RT-PCR showing the human β^41/42 mutation gene was expressed in MEL cells.Conclusions: An in vitro cell model capable of highly expression of human β^41/42 mutation gene in MEL cells was established successfully.This may be a valuable gene therapy model for human β thalassemia mutation β^41/42.