Prevention of Transcriptional γ-globin Gene Silencing by Inducing The Hereditary Persistence of Fetal Hemoglobin Point Mutation Using Chimeraplast-Mediated Gene Targeting
Journal Title: Cell Journal(Yakhteh) - Year 2018, Vol 20, Issue 3
Abstract
Objective: Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in HBG promoter in prevention of transcriptional silencing of the γ-globin. Materials and Methods: In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively. Results: Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells (EPCs) were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in β/γ-globin gene expression in transfected EPCs at the experimental end point (day 28) whereas, due to 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. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.
Authors and Affiliations
Reza Ranjbaran, Mahin Nikogoftar Zarif, Sedigheh Sharifzadeh, Habibollah Golafshan, Ali Akbar Pourfathollah
Keywords
Related Articles
- EP ID EP469152
- DOI 10.22074/ cellj.2018.5181
- Views 203
- Downloads 0
Regulated Acyl-CoA Synthetase Short-Chain Family Member 2 Accumulation during Spermatogenesis
Objective: Acyl-CoA synthetase short-chain family member 2 (ACSS2) activity provides a major source of acetyl-CoA to drive histone acetylation. This study aimed to unravel the ACSS2 expression during mouse spermatogenesi...
Conditioned Media Derived from Human Adipose Tissue Mesenchymal Stromal Cells Improves Primary Hepatocyte Maintenance
Objective: Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functiona...
A Homozygous 1.16 Megabases Microdeletion At 8p22 Including The Whole TUSC3 In A Three Years Old Girl With Intellectual Disability And Speech Delay
Intellectual disability (ID) is defined as an intelligence quotient (IQ) level below than 70. In the present paper, a 1.16 megabases (Mb) homozygous deletion in the 8p22 region was identified in a three years old girl wi...
A Pathogenic Homozygous Mutation in The Pleckstrin Homology Domain of RASA1 Is Responsible for Familial Tricuspid Atresia in An Iranian Consanguineous Family
Objective: Tricuspid atresia (TA) is a rare life-threatening form of congenital heart defect (CHD). The genetic mechanisms underlying TA are not clearly understood. According to previous studies, the endocardial cushioni...
Histological Evidence for Therapeutic Induction of Angiogenesis Using Mast Cells and Platelet-Rich Plasma within A Bioengineered Scaffold following Rat Hindlimb Ischemia
Objective: Peripheral arterial disease results from obstructed blood flow in arteries and increases the risk of amputation in acute cases. Therapeutic angiogenesis using bioengineered tissues composed of a chitosan scaff...