A method to estimate the efficiency of gene expression from an integrated retroviral vector

<p>Abstract</p> <p>Background</p> <p>Proviral gene expression is a critical step in the retroviral life cycle and an important determinant in the efficiency of retrovirus based gene therapy vectors. There is as yet no method described that can assess the efficiency of p...

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Main Authors: Lever Andrew, Mok Hoi
Format: Article
Language:English
Published: BMC 2006-08-01
Series:Retrovirology
Online Access:http://www.retrovirology.com/content/3/1/51
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author Lever Andrew
Mok Hoi
author_facet Lever Andrew
Mok Hoi
author_sort Lever Andrew
collection DOAJ
description <p>Abstract</p> <p>Background</p> <p>Proviral gene expression is a critical step in the retroviral life cycle and an important determinant in the efficiency of retrovirus based gene therapy vectors. There is as yet no method described that can assess the efficiency of proviral gene expression while vigorously excluding the contribution from unstable species such as passively transferred plasmid and LTR circles. Here, we present a method that can achieve this.</p> <p>Results</p> <p>Proviral gene expression was detected by the activity of the puromycin resistance gene encoded in the viral vector, and quantified by comparing the growth curve of the sample under puromycin selection to that of a series of calibration cultures. Reproducible estimates of the efficiency of proviral gene expression could be derived. We confirm that contamination from unstable species such as passively transferred plasmid used in viral vector production and unintegrated viral DNA can seriously confound estimates of the efficiency of transduction. This can be overcome using a PCR based on limiting dilution analysis.</p> <p>Conclusion</p> <p>A simple, low cost method was developed that should be useful in studying the biology of retroviruses and for the development of expression systems for retrovirus based gene therapy.</p>
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spelling doaj.art-fb5a6bcf17f54f818a459b392812f51c2022-12-21T23:13:33ZengBMCRetrovirology1742-46902006-08-01315110.1186/1742-4690-3-51A method to estimate the efficiency of gene expression from an integrated retroviral vectorLever AndrewMok Hoi<p>Abstract</p> <p>Background</p> <p>Proviral gene expression is a critical step in the retroviral life cycle and an important determinant in the efficiency of retrovirus based gene therapy vectors. There is as yet no method described that can assess the efficiency of proviral gene expression while vigorously excluding the contribution from unstable species such as passively transferred plasmid and LTR circles. Here, we present a method that can achieve this.</p> <p>Results</p> <p>Proviral gene expression was detected by the activity of the puromycin resistance gene encoded in the viral vector, and quantified by comparing the growth curve of the sample under puromycin selection to that of a series of calibration cultures. Reproducible estimates of the efficiency of proviral gene expression could be derived. We confirm that contamination from unstable species such as passively transferred plasmid used in viral vector production and unintegrated viral DNA can seriously confound estimates of the efficiency of transduction. This can be overcome using a PCR based on limiting dilution analysis.</p> <p>Conclusion</p> <p>A simple, low cost method was developed that should be useful in studying the biology of retroviruses and for the development of expression systems for retrovirus based gene therapy.</p>http://www.retrovirology.com/content/3/1/51
spellingShingle Lever Andrew
Mok Hoi
A method to estimate the efficiency of gene expression from an integrated retroviral vector
Retrovirology
title A method to estimate the efficiency of gene expression from an integrated retroviral vector
title_full A method to estimate the efficiency of gene expression from an integrated retroviral vector
title_fullStr A method to estimate the efficiency of gene expression from an integrated retroviral vector
title_full_unstemmed A method to estimate the efficiency of gene expression from an integrated retroviral vector
title_short A method to estimate the efficiency of gene expression from an integrated retroviral vector
title_sort method to estimate the efficiency of gene expression from an integrated retroviral vector
url http://www.retrovirology.com/content/3/1/51
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AT mokhoi amethodtoestimatetheefficiencyofgeneexpressionfromanintegratedretroviralvector
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AT mokhoi methodtoestimatetheefficiencyofgeneexpressionfromanintegratedretroviralvector