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An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus
BACKGROUND: A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance effic...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939624/ https://www.ncbi.nlm.nih.gov/pubmed/27403084 http://dx.doi.org/10.1186/s12575-016-0044-z |
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author | Sengupta, Ranjita Mukherjee, Chandreyee Sarkar, Nandita Sun, Zhihong Lesnik, Jacob Huang, Joseph Lu, Biao |
author_facet | Sengupta, Ranjita Mukherjee, Chandreyee Sarkar, Nandita Sun, Zhihong Lesnik, Jacob Huang, Joseph Lu, Biao |
author_sort | Sengupta, Ranjita |
collection | PubMed |
description | BACKGROUND: A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done. RESULTS: Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed. CONCLUSIONS: An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12575-016-0044-z) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4939624 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-49396242016-07-12 An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus Sengupta, Ranjita Mukherjee, Chandreyee Sarkar, Nandita Sun, Zhihong Lesnik, Jacob Huang, Joseph Lu, Biao Biol Proced Online Methodology BACKGROUND: A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done. RESULTS: Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed. CONCLUSIONS: An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12575-016-0044-z) contains supplementary material, which is available to authorized users. BioMed Central 2016-07-11 /pmc/articles/PMC4939624/ /pubmed/27403084 http://dx.doi.org/10.1186/s12575-016-0044-z Text en © The Author(s). 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Sengupta, Ranjita Mukherjee, Chandreyee Sarkar, Nandita Sun, Zhihong Lesnik, Jacob Huang, Joseph Lu, Biao An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus |
title | An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus |
title_full | An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus |
title_fullStr | An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus |
title_full_unstemmed | An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus |
title_short | An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus |
title_sort | optimized protocol for packaging pseudotyped integrase defective lentivirus |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939624/ https://www.ncbi.nlm.nih.gov/pubmed/27403084 http://dx.doi.org/10.1186/s12575-016-0044-z |
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