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Evaluation of knocking down of the RNAi mediated gamma globin repressor into K562 cells, for gene therapy of beta-thalassemia
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Ali Mohammad Asgharian *   , Mehdi Banan , Hossein Najmabadi |
| Instructor, Department of Cell and Molecular Biology, Tonekabone Branch, Islamic Azad University, Tonekabone, Iran , mehranasgharian@yahoo.com |
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Abstract: (9770 Views) |
| Background and Aim: Beta-thalassemia is a genetic disorder manifested by the presence of anemia in adult patients. One approach to treatment of beta-thalassemia is induction of the fetal gamma-globin gene. One of the gamma-globin repressors is a complex called DRED (Direct repeat erythroid-definitive). DRED is composed of TR2 and TR4 DNA binding subunits. The aim of this study was to set up the RNAi system to increase the expression of the gamma-globin gene. Materials and Methods: In this study, lipofectaminTM2000 was used to transfect siRNA molecules and pSV-β-Galactosidase vector was used as a reporter to monitor transfection efficiency. Real-time PCR method was used to measure TR4 knockdown and gamma-globin expression levels. Results: Our findings showed that K562 cells were transfected by 40% using LipofectamineTM 2000. Also, the level of TR4 expression decreased by 44% after using TR4siRNA, even though the expression of gamma-globin gene was induced by 1.18 times. Conclusion: Despite a 44% knockdown of TR4, no increase in gamma-globin mRNA was observed. Two factors may count for this observation first, TR4 knockdown may have been limited by our transfection efficiency. Second, simultaneous knockdown of TR2 and TR4 may lead to increased gamma-globin levels. In conclusion, although knocking down of TR4 expression occurred by using RNAi system, this can not be a solitary efficient way to induce the gamma-globin expression. |
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| Keywords: beta-Thalassemia, TR4 siRNA, TR4 inhibition, gamma-Globins, K562 Cells |
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Full-Text [PDF 329 kb]
(3160 Downloads)
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Type of Study: Original |
Subject:
Medicine
Received: 2017 | Accepted: 2017 | Published: 2017
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