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Gene manipulation of human adipose-derived mesenchymal stem cells by miR-34a
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Katayoun Bahman Soufiani1    , Ali Akbar Pourfathollah 2, Mahin Nikougoftar Zarif3 , Ehsan Arefian4 |
1- PhD Candidate, Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2- Assistant Professor, Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran , pourfa@modares.ac.ir
3- Associate Professor, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
4- Assistant Professor, Molecular Virology Lab, Department of Microbiology, School of Biology, college of Science, University of Tehran, Tehran, Iran |
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Abstract: (2391 Views) |
Background: Safe and effective gene therapy is considered as one of the therapeutic goals in many diseases. Due to the important role of stem cells in cell therapy, this study aimed to produce human adipose-derived mesenchymal stem cells (hASCs) using the miR-34a overexpression.
Materials and methods: The hsa-mir-34a precursor sequence was cloned into the PCDH lentiviral vector. The recombinant vector and two helper vectors, i.e. psPAX and pMD2, were transferred into HEK-293T cell line by calcium phosphate method. Viral supernatant was collected and concentrated by ultracentrifuge. On the fourth day, transduced HEK-293 T cells were analyzed by flowcytometry. After the determination of viral concentration, hASC cells were transduced with condensed viruses. RNA extraction and cDNA synthesis were performed in order to assess miR-34a expression level by Real Time PCR.
Results: The hsa-mir-34a precursor sequence cloned into PCDH vector was confirmed by colony-PCR and DNA sequencing. Transduction of HEK-293T cells and hASCs were confirmed under fluorescent inverted microscope and flowcytometry. The assessment of miR-34a expression level in infected cells with recombinant virus showed that the expression ratio of miR-34a in the test group was significantly higher than the control group (P=0.001).
Conclusion: This study showed that lentiviral systems can be used to insert actopic genes like miR-34a into cells. It also showed that genetically manipulated stem cells can be used as a delivery system to deliver miR-34a or other genes. |
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| Keywords: Stem cells, Lentivirus, Transduction, Transfection, HEK-293T, miR-34a. |
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Full-Text [PDF 633 kb]
(1404 Downloads)
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Semi-pilot: Basic |
Subject:
Molecular Biology
Received: 2020/05/11 | Accepted: 2020/09/5 | Published: 2021/03/24
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