Abstract
The severity of β-thalassemia syndrome is associated with precipitation of the cytotoxic excessive free α-hemoglobin. We have established a therapy strategy aiming at stabilizing excessive free α-hemoglobin by decreasing its precipitation using erythroid differentiation-related factor (EDRF). To identify the functional EDRF promoter, different length of human EDRF promoter sequence was cloned upstream of green fluorescence protein (GFP) to drive GFP expression. After transfection, the intensity of GFP expression was monitored by microscopy and fluorescence activated cell sorter analysis. The 372 base pair sequence (−116∼+256 bp) was found to be most effective to induce the GFP expression and thereby cloned into pcDNA-EDRF vector to drive EDRF expression. After introducing pcDNA-EDRF vector into MEL cells and healthy mice, we confirmed high EDRF expression at both mRNA and protein levels using reverse transcription polymerase chain reacion and enzyme-linked immunosorbent assay. We further tested the function of EDRF overexpression in β-thalassemia mice by pcDNA-EDRF injection. One week later after treatment, the hemoglobin value, poikilocytosis and target cells of β-thalassemia mice were found to be ameliorated obviously. These results indicated that EDRF is a potential therapeutic target by improving hematological parameters of β-thalassemia.
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Abbreviations
- AHSP:
-
α-hemoglobin stabilizing protein
- bp:
-
base pairs
- cDNA:
-
complementary DNA
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- DMSO:
-
dimethylsulfoxide
- EDRF:
-
erythroid differentiation-related factor
- ELISA:
-
enzymelinked immunosorbent assay
- FACS:
-
fluorescence activated cell sorter
- GFP:
-
green fluorescence protein
- Hbbth-4/Hbb+ :
-
β-thalassemia heterozygote
- MEL:
-
mouse erythroleukemia
- NIH3T3:
-
mouse embryonic fibroblast cells
- RT-PCR:
-
reverse transcription polymerase chain reaction
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Wang, P., Xie, S., Chi, Y. et al. Erythroid differentiation-related factor expression driven by effective promoter to ameliorate the anemia syndrome. Biologia 64, 826–832 (2009). https://doi.org/10.2478/s11756-009-0141-4
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DOI: https://doi.org/10.2478/s11756-009-0141-4