Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia
Objective(s): β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites t...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Mashhad University of Medical Sciences
2016-10-01
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| Series: | Iranian Journal of Basic Medical Sciences |
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| Online Access: | http://ijbms.mums.ac.ir/article_7731_bf7a1207e8bbca99d681d259846d4e30.pdf |
| _version_ | 1811298244756504576 |
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| author | Nasrin Heydari Laleh Shariati Hossein Khanahmad Zahra Hejazi Mansoureh Shahbazi Mansoor Salehi |
| author_facet | Nasrin Heydari Laleh Shariati Hossein Khanahmad Zahra Hejazi Mansoureh Shahbazi Mansoor Salehi |
| author_sort | Nasrin Heydari |
| collection | DOAJ |
| description | Objective(s): β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites to the K562 cell line. Materials and Methods: A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on β-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 μg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 µg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells. Results: A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones. Conclusion: The findings demonstrate that the spongy effect of KLF1-binding site on BCL11A and β-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease. |
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| format | Article |
| id | doaj.art-b86befbb267d47549d8fa4137bd270bb |
| institution | Directory Open Access Journal |
| issn | 2008-3866 2008-3874 |
| language | English |
| last_indexed | 2024-04-13T06:16:33Z |
| publishDate | 2016-10-01 |
| publisher | Mashhad University of Medical Sciences |
| record_format | Article |
| series | Iranian Journal of Basic Medical Sciences |
| spelling | doaj.art-b86befbb267d47549d8fa4137bd270bb2022-12-22T02:58:48ZengMashhad University of Medical SciencesIranian Journal of Basic Medical Sciences2008-38662008-38742016-10-0119101063106910.22038/ijbms.2016.77317731Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemiaNasrin Heydari0Laleh Shariati1Hossein Khanahmad2Zahra Hejazi3Mansoureh Shahbazi4Mansoor Salehi5Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IranDepartment of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IranDepartment of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran|Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable Disease, Isfahan University of Medical Sciences, Isfahan, IranDepartment of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IranDepartment of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IranDepartment of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IranObjective(s): β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites to the K562 cell line. Materials and Methods: A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on β-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 μg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 µg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells. Results: A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones. Conclusion: The findings demonstrate that the spongy effect of KLF1-binding site on BCL11A and β-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease.http://ijbms.mums.ac.ir/article_7731_bf7a1207e8bbca99d681d259846d4e30.pdfHb F inductionKLF1 geneβ-switchingβ-thalassemiaγ-globin |
| spellingShingle | Nasrin Heydari Laleh Shariati Hossein Khanahmad Zahra Hejazi Mansoureh Shahbazi Mansoor Salehi Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia Iranian Journal of Basic Medical Sciences Hb F induction KLF1 gene β-switching β-thalassemia γ-globin |
| title | Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia |
| title_full | Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia |
| title_fullStr | Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia |
| title_full_unstemmed | Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia |
| title_short | Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia |
| title_sort | gamma reactivation using the spongy effect of klf1 binding site sequence an approach in gene therapy for beta thalassemia |
| topic | Hb F induction KLF1 gene β-switching β-thalassemia γ-globin |
| url | http://ijbms.mums.ac.ir/article_7731_bf7a1207e8bbca99d681d259846d4e30.pdf |
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