Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis

Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis

In this study, we are taking step to actively manage osteoarthritis that may help gain control over osteoarthritic pain and delay the degenerative changes in articular cartilage in future. We transiently over expressed cartilage transcriptional factor, human sox-9 gene in chondrocytes derived from c...

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Main Authors: Sha'ban, Munirah, Osman Cassim, Samsudin, Mohd Yahya, Nor Hamdan, Saim, Aminuddin, Idrus, Ruszymah
Format: Article
Language:English
Published: Korean Tissue Engineering and Regenerative Medicine Society 2011
Subjects:
R Medicine (General)
Online Access:http://irep.iium.edu.my/21274/
http://irep.iium.edu.my/21274/
http://irep.iium.edu.my/21274/1/Sox-9_Transient_Transfection_Enhances_Chondrogenic_Expression_of_Osteoarthritic_Human_Articular.pdf
id iium-21274
recordtype eprints
spelling iium-212742014-06-04T07:50:32Z http://irep.iium.edu.my/21274/ Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis Sha'ban, Munirah Osman Cassim, Samsudin Mohd Yahya, Nor Hamdan Saim, Aminuddin Idrus, Ruszymah R Medicine (General) In this study, we are taking step to actively manage osteoarthritis that may help gain control over osteoarthritic pain and delay the degenerative changes in articular cartilage in future. We transiently over expressed cartilage transcriptional factor, human sox-9 gene in chondrocytes derived from consented osteoarthritic patients after joint surgery. The expression vector carrying human sox-9 gene, pAdTrack-sox9 was transformed into One Shot TOP 10 Chemically Competent E. coli according to the manufacturer protocol. Plasmid purification was performed in accordance with QIAGEN (R) plasmid purification kit procedure. We compared the efficiency between two transfection techniques i.e. lipofection using Lipofectamine (TM) 2000 kit from Invitrogen, USA and nucleofection using Human Chondrocytes Nucleofector (R) kit from Amaxa Biosystem, Germany. Chondrocytes were cultured and transfected with sox-9 gene at passage 1 according to the manufacturers' protocols. Transfected chondrocytes were expanded until passage 3. Expression of chondrogenic markers namely collagen type H, aggrecan core protein and sox-9 were evaluated by quantitative RT-PCR method using iScript (TM) One Step RT-PCR Kit with SYBR Green, BIO-RAD. Chondrogenic dedifferentiation marker, collagen type I was also analyzed using the quantitative RT-PCR method. Expression level of each targeted gene was normalized to the housekeeping gene, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Overall efficiency ranging from 50% to 60% could be achieved using both transfection techniques. Transiently transfecting cells demonstrated remarkable competency sustaining specific chondrogenic genes namely collagen type H, aggrecan core protein and sox-9, significantly better than in the non-transfected cells. It is believed that this preliminary finding has to be extended to develop its full potential since sox-9 transcription factor is essential for chondrocyte differentiation and cartilage formation. Sox9 gene therapy would delay the degenerative changes in articular cartilage which is consistent to the up-regulation of cartilage-specific markers especially collagen type II synthesis in vivo. Korean Tissue Engineering and Regenerative Medicine Society 2011 Article PeerReviewed application/pdf en http://irep.iium.edu.my/21274/1/Sox-9_Transient_Transfection_Enhances_Chondrogenic_Expression_of_Osteoarthritic_Human_Articular.pdf Sha'ban, Munirah and Osman Cassim, Samsudin and Mohd Yahya, Nor Hamdan and Saim, Aminuddin and Idrus, Ruszymah (2011) Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis. Tissue Engineering and Regenerative Medicine, 8 (1). pp. 32-41. ISSN 2212-5469 (O), 1738-2696 (P) http://www.kterms.or.kr/
repository_type Digital Repository
institution_category Local University
institution International Islamic University Malaysia
building IIUM Repository
collection Online Access
language English
topic R Medicine (General)
spellingShingle R Medicine (General)
Sha'ban, Munirah
Osman Cassim, Samsudin
Mohd Yahya, Nor Hamdan
Saim, Aminuddin
Idrus, Ruszymah
Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis
description In this study, we are taking step to actively manage osteoarthritis that may help gain control over osteoarthritic pain and delay the degenerative changes in articular cartilage in future. We transiently over expressed cartilage transcriptional factor, human sox-9 gene in chondrocytes derived from consented osteoarthritic patients after joint surgery. The expression vector carrying human sox-9 gene, pAdTrack-sox9 was transformed into One Shot TOP 10 Chemically Competent E. coli according to the manufacturer protocol. Plasmid purification was performed in accordance with QIAGEN (R) plasmid purification kit procedure. We compared the efficiency between two transfection techniques i.e. lipofection using Lipofectamine (TM) 2000 kit from Invitrogen, USA and nucleofection using Human Chondrocytes Nucleofector (R) kit from Amaxa Biosystem, Germany. Chondrocytes were cultured and transfected with sox-9 gene at passage 1 according to the manufacturers' protocols. Transfected chondrocytes were expanded until passage 3. Expression of chondrogenic markers namely collagen type H, aggrecan core protein and sox-9 were evaluated by quantitative RT-PCR method using iScript (TM) One Step RT-PCR Kit with SYBR Green, BIO-RAD. Chondrogenic dedifferentiation marker, collagen type I was also analyzed using the quantitative RT-PCR method. Expression level of each targeted gene was normalized to the housekeeping gene, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Overall efficiency ranging from 50% to 60% could be achieved using both transfection techniques. Transiently transfecting cells demonstrated remarkable competency sustaining specific chondrogenic genes namely collagen type H, aggrecan core protein and sox-9, significantly better than in the non-transfected cells. It is believed that this preliminary finding has to be extended to develop its full potential since sox-9 transcription factor is essential for chondrocyte differentiation and cartilage formation. Sox9 gene therapy would delay the degenerative changes in articular cartilage which is consistent to the up-regulation of cartilage-specific markers especially collagen type II synthesis in vivo.
format Article
author Sha'ban, Munirah
Osman Cassim, Samsudin
Mohd Yahya, Nor Hamdan
Saim, Aminuddin
Idrus, Ruszymah
author_facet Sha'ban, Munirah
Osman Cassim, Samsudin
Mohd Yahya, Nor Hamdan
Saim, Aminuddin
Idrus, Ruszymah
author_sort Sha'ban, Munirah
title Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis
title_short Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis
title_full Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis
title_fullStr Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis
title_full_unstemmed Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis
title_sort sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis
publisher Korean Tissue Engineering and Regenerative Medicine Society
publishDate 2011
url http://irep.iium.edu.my/21274/
http://irep.iium.edu.my/21274/
http://irep.iium.edu.my/21274/1/Sox-9_Transient_Transfection_Enhances_Chondrogenic_Expression_of_Osteoarthritic_Human_Articular.pdf
first_indexed 2023-09-18T20:32:15Z
last_indexed 2023-09-18T20:32:15Z
_version_ 1777408814352433152

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