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Cell Journal، جلد ۲۰، شماره ۳، صفحات ۳۱۸-۳۲۵
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عنوان انگلیسی Prevention of Transcriptional γ-globin Gene Silencing by Inducing The Hereditary Persistence of Fetal Hemoglobin Point Mutation Using Chimeraplast-Mediated Gene Targeting
چکیده انگلیسی مقاله Objective: Hemoglobin F (HbF) augmentation is considered as a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing inspired by hereditary persistence of fetal hemoglobin (HPFH) genetic pattern might be a suitable strategy for increment of HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in γ-globin gene promoter in prevention of transcriptional silencing of γ-globin gene. Materials and methods: In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and k562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology assessment and flow cytometric analysis of CD235a expression. A synthesized chimeraplast was designed and transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was evaluated by flow cytometry. PCR-RFLP and DNA sequencing verified the incidence of site-directed mutagenesis. Gene conversion frequency was quantified through AS-qPCR and globin genes expression was assessed by qRT-PCR. Results: Increase in CD235a expressing cells and the observation made for different stages of erythroid maturation over time confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on day 18-21 of HSCs differentiation. Flow cytometric analysis revealed that more than 70% of EPCs were transfected with chimeraplast. The highest gene conversion efficiency was 7.2% and 11.1% in EPCs and K562 cells respectively. The induced mutation led to 1.97-fold decrease in β/γ globin gene expression in transfected EPCs at the experimental end point (day 28) whereas because of the absence of β-globin gene expression following K562 differentiation, this rate was not evaluable. Conclusion: Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and k562 cells. It also demonstrated that stimulated point mutation was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.
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نویسندگان مقاله | Reza Ranjbaran


| Mahin Nikogoftar Zarif


| Sedigheh Sharifzadeh


| Habibollah Golafshan


| Ali Akbar Pourfathollah


نشانی اینترنتی http://celljournal.org/journal/article/abstract/5181
فایل مقاله اشکال در دسترسی به فایل - ./files/site1/rds_journals/16/article-16-851776.pdf
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