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Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro
Gene regulation remains one of the major challenges for gene therapy in clinical trials. In the present study, we first generated a binary tetracycline-on (Tet-On) system based on two lentivirus vectors, one expressing both human glial cell line-derived neurotrophic factor (hGDNF) and humanized reco...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661514/ https://www.ncbi.nlm.nih.gov/pubmed/23717608 http://dx.doi.org/10.1371/journal.pone.0064389 |
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author | Yang, Wei-Hua Yang, Chun Xue, Yue-Qiang Lu, Tao Reiser, Jakob Zhao, Li-Ru Duan, Wei-Ming |
author_facet | Yang, Wei-Hua Yang, Chun Xue, Yue-Qiang Lu, Tao Reiser, Jakob Zhao, Li-Ru Duan, Wei-Ming |
author_sort | Yang, Wei-Hua |
collection | PubMed |
description | Gene regulation remains one of the major challenges for gene therapy in clinical trials. In the present study, we first generated a binary tetracycline-on (Tet-On) system based on two lentivirus vectors, one expressing both human glial cell line-derived neurotrophic factor (hGDNF) and humanized recombinant green fluorescent protein (hrGFP) genes under second-generation tetracycline response element (TRE), and the other expressing the advanced reverse tetracycline-controlled transactivator - rtTA2S-M2 under a human minimal cytomegalovirus immediate early (CMV-IE) promoter. This system allows simultaneous expression of hGDNF and hrGFP genes in the presence of doxycycline (Dox). Human bone marrow-derived mesenchymal stem cells (hMSCs) were transduced with the binary Tet-On lentivirus vectors and characterized in vitro in the presence (On) or absence (Off) of Dox. The expression of hGDNF and hrGFP transgenes in transduced hMSCs was tightly regulated as determined by flow cytometry (FCM), GDNF enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR). There was a dose-dependent regulation for hrGFP transgene expression. The levels of hGDNF protein in culture medium were correlated with the mean fluorescence intensity (MFI) units of hrGFP. The levels of transgene background expression were very low in the absence of Dox. The treatment of the conditioned medium from cultures of transduced hMSCs in the presence of Dox protected SH-SY5Y cells against 6-hydroxydopamine (6-OHDA) toxicity as determined by cell viability using 3, [4,5-dimethylthiazol-2-yl]- diphenyltetrazolium bromide (MTT) assay. The treatment of the conditioned medium was also found to improve the survival of dopaminergic (DA) neurons of ventral mesencephalic (VM) tissue in serum-free culture conditions as assessed by cell body area, the number of neurites and dendrite branching points, and proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) cells. Our inducible lentivirus-mediated hGDNF gene delivery system may provide useful tools for basic research on gene therapy for chronic neurological disorders such as Parkinson’s disease (PD). |
format | Online Article Text |
id | pubmed-3661514 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-36615142013-05-28 Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro Yang, Wei-Hua Yang, Chun Xue, Yue-Qiang Lu, Tao Reiser, Jakob Zhao, Li-Ru Duan, Wei-Ming PLoS One Research Article Gene regulation remains one of the major challenges for gene therapy in clinical trials. In the present study, we first generated a binary tetracycline-on (Tet-On) system based on two lentivirus vectors, one expressing both human glial cell line-derived neurotrophic factor (hGDNF) and humanized recombinant green fluorescent protein (hrGFP) genes under second-generation tetracycline response element (TRE), and the other expressing the advanced reverse tetracycline-controlled transactivator - rtTA2S-M2 under a human minimal cytomegalovirus immediate early (CMV-IE) promoter. This system allows simultaneous expression of hGDNF and hrGFP genes in the presence of doxycycline (Dox). Human bone marrow-derived mesenchymal stem cells (hMSCs) were transduced with the binary Tet-On lentivirus vectors and characterized in vitro in the presence (On) or absence (Off) of Dox. The expression of hGDNF and hrGFP transgenes in transduced hMSCs was tightly regulated as determined by flow cytometry (FCM), GDNF enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR). There was a dose-dependent regulation for hrGFP transgene expression. The levels of hGDNF protein in culture medium were correlated with the mean fluorescence intensity (MFI) units of hrGFP. The levels of transgene background expression were very low in the absence of Dox. The treatment of the conditioned medium from cultures of transduced hMSCs in the presence of Dox protected SH-SY5Y cells against 6-hydroxydopamine (6-OHDA) toxicity as determined by cell viability using 3, [4,5-dimethylthiazol-2-yl]- diphenyltetrazolium bromide (MTT) assay. The treatment of the conditioned medium was also found to improve the survival of dopaminergic (DA) neurons of ventral mesencephalic (VM) tissue in serum-free culture conditions as assessed by cell body area, the number of neurites and dendrite branching points, and proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) cells. Our inducible lentivirus-mediated hGDNF gene delivery system may provide useful tools for basic research on gene therapy for chronic neurological disorders such as Parkinson’s disease (PD). Public Library of Science 2013-05-22 /pmc/articles/PMC3661514/ /pubmed/23717608 http://dx.doi.org/10.1371/journal.pone.0064389 Text en © 2013 Yang et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Yang, Wei-Hua Yang, Chun Xue, Yue-Qiang Lu, Tao Reiser, Jakob Zhao, Li-Ru Duan, Wei-Ming Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro |
title | Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro
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title_full | Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro
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title_fullStr | Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro
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title_full_unstemmed | Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro
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title_short | Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro
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title_sort | regulated expression of lentivirus-mediated gdnf in human bone marrow-derived mesenchymal stem cells and its neuroprotection on dopaminergic cells in vitro |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661514/ https://www.ncbi.nlm.nih.gov/pubmed/23717608 http://dx.doi.org/10.1371/journal.pone.0064389 |
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