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Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer

Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike non-viral methods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome, which can lead to long-term transgene expression. Lentiviruses have advantages over oth...

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Main Authors: Ngai, Siew Ching, Rajesh, Ramasamy, Abdullah, Syahril
Format: Article
Language:English
Published: Universiti Putra Malaysia Press 2012
Online Access:http://psasir.upm.edu.my/id/eprint/40462/1/13.%20Production%20of%20Lentivirus%20Carrying%20Green%20Fluorescent%20Protein%20with.pdf
http://psasir.upm.edu.my/id/eprint/40462/
http://www.pertanika.upm.edu.my/Pertanika%20PAPERS/JST%20Vol.%2020%20%282%29%20Jul.%202012/06%20Pg%20269-281.pdf
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spelling my.upm.eprints.404622015-11-04T03:26:42Z http://psasir.upm.edu.my/id/eprint/40462/ Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer Ngai, Siew Ching Rajesh, Ramasamy Abdullah, Syahril Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike non-viral methods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome, which can lead to long-term transgene expression. Lentiviruses have advantages over other types of viruses due to their capacity to transduce non-dividing cells. An optimized generation of lentiviruses carrying green fluorescent protein (GFP) reporter gene driven by either UbC (LV/UbC/GFP) or CMV (LV/CMV/GFP) promoter is described in this paper. The lentiviruses were produced by co-transfecting lentiviral expression constructs and packaging mix into 293FT lentivirus producer cell lines. Lipofectamine was highly efficient in transfecting the cells compared to Transfast and Polyethyleneimine (PEI). Following cell transfection, syncytia were clearly visible at day 2. Lentiviruses were harvested at days 1, 2 and 3 post-transfection. The highest transduction efficiency was read from LV/CMV/GFP harvested at day 2 post-transfection and LV/UbC/GFP harvested at day 3 post-transfection. Finally, the GFP expression in COS-7 cells was determined at day 2 and day 14 post-transduction for transient and stable GFP expression. It was found that the GFP expression declined overtime. However, the transduction efficiency and duration of the transgene expression in COS-7 cells transduced with LV/CMV/GFP were higher compared to LV/UbC/GFP. In conclusion, we have successfully produced lentiviruses carrying GFP with different promoters and shown that the viruses were able to infect COS-7 cells at different efficiencies. Meanwhile, the generation of the active lentiviruses will allow us to the subsequent analysis of the effect of regulatory elements in future study. Universiti Putra Malaysia Press 2012 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/40462/1/13.%20Production%20of%20Lentivirus%20Carrying%20Green%20Fluorescent%20Protein%20with.pdf Ngai, Siew Ching and Rajesh, Ramasamy and Abdullah, Syahril (2012) Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer. Pertanika Journal of Science & Technology, 20 (2). pp. 269-281. ISSN 0128-7680; ESSN: 2231-8526 http://www.pertanika.upm.edu.my/Pertanika%20PAPERS/JST%20Vol.%2020%20%282%29%20Jul.%202012/06%20Pg%20269-281.pdf
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike non-viral methods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome, which can lead to long-term transgene expression. Lentiviruses have advantages over other types of viruses due to their capacity to transduce non-dividing cells. An optimized generation of lentiviruses carrying green fluorescent protein (GFP) reporter gene driven by either UbC (LV/UbC/GFP) or CMV (LV/CMV/GFP) promoter is described in this paper. The lentiviruses were produced by co-transfecting lentiviral expression constructs and packaging mix into 293FT lentivirus producer cell lines. Lipofectamine was highly efficient in transfecting the cells compared to Transfast and Polyethyleneimine (PEI). Following cell transfection, syncytia were clearly visible at day 2. Lentiviruses were harvested at days 1, 2 and 3 post-transfection. The highest transduction efficiency was read from LV/CMV/GFP harvested at day 2 post-transfection and LV/UbC/GFP harvested at day 3 post-transfection. Finally, the GFP expression in COS-7 cells was determined at day 2 and day 14 post-transduction for transient and stable GFP expression. It was found that the GFP expression declined overtime. However, the transduction efficiency and duration of the transgene expression in COS-7 cells transduced with LV/CMV/GFP were higher compared to LV/UbC/GFP. In conclusion, we have successfully produced lentiviruses carrying GFP with different promoters and shown that the viruses were able to infect COS-7 cells at different efficiencies. Meanwhile, the generation of the active lentiviruses will allow us to the subsequent analysis of the effect of regulatory elements in future study.
format Article
author Ngai, Siew Ching
Rajesh, Ramasamy
Abdullah, Syahril
spellingShingle Ngai, Siew Ching
Rajesh, Ramasamy
Abdullah, Syahril
Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
author_facet Ngai, Siew Ching
Rajesh, Ramasamy
Abdullah, Syahril
author_sort Ngai, Siew Ching
title Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
title_short Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
title_full Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
title_fullStr Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
title_full_unstemmed Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
title_sort production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
publisher Universiti Putra Malaysia Press
publishDate 2012
url http://psasir.upm.edu.my/id/eprint/40462/1/13.%20Production%20of%20Lentivirus%20Carrying%20Green%20Fluorescent%20Protein%20with.pdf
http://psasir.upm.edu.my/id/eprint/40462/
http://www.pertanika.upm.edu.my/Pertanika%20PAPERS/JST%20Vol.%2020%20%282%29%20Jul.%202012/06%20Pg%20269-281.pdf
_version_ 1643832723032244224
score 13.211869

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