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Thalassemia Reports
Volume 4
Issue 2
10.4081/thal.2014.2189
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Thalassemia Reports is published by MDPI from Volume 12 Issue 1 (2022). Previous articles were published by another publisher in Open Access under a CC-BY (or CC-BY-NC-ND) licence, and they are hosted by MDPI on mdpi.com as a courtesy and upon agreement with PAGEPress.
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Article

Development of Plasmids for Quantitative Detection of Integrated Lentiviral Vectors and Evaluation of Culture Time to Perform Vector Titer by Real-Time Quantitative Polymerase Chain Reaction Assay

by
Elena Baiamonte
,
Mariella Bagliesi
,
Valentina Motta
,
Barbara Spina
and
Alice Pecoraro
*
Alice Pecoraro, UOC Ematologia per le Malattie Rare delSangue e degli Organi Ematopoietici, Dipartimento di Oncologia edEmatologia, AO Ospedali Riuniti VillaSofia-Cervello, 90146 Palermo, Italy
*
Author to whom correspondence should be addressed.
Thalass. Rep. 2014, 4(2), 2189; https://doi.org/10.4081/thal.2014.2189
Submission received: 9 December 2013 / Revised: 17 February 2014 / Accepted: 13 March 2014 / Published: 29 September 2014

Abstract

The accurate assessment of provirus copy number per cell (VCN/cell) is a fundamental issue in transgenesis as well as in gene therapy studies based on stably integrated vectors. To this end, real-time quantitative polymerase chain reaction (qPCR) is a powerful method but it is sensible to differences in quality or concentration of the two-plasmid preparations used for the construction of the standard curves. In order to minimize technical errors we included genome specific sequences (mouse or human) and vector specific sequences in the same plasmid. We evaluated the specificity and sensitivity of these bivalent plasmids by qPCR analysis on mouse and human genomic DNA containing a known number of a reporter lentiviral vector and we found that the system is reliable to measure up to 0.1 VCN/cell. Here we have applied this assay to measure vector titer of virus stock preparations and to determine the optimal cell passages at which viral titration effectively reflects the number of integrated vectors.
Keywords: vector copy number; real-time quantitative polymerase chain reaction; lentiviral vector titer vector copy number; real-time quantitative polymerase chain reaction; lentiviral vector titer

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MDPI and ACS Style

Baiamonte, E.; Bagliesi, M.; Motta, V.; Spina, B.; Pecoraro, A. Development of Plasmids for Quantitative Detection of Integrated Lentiviral Vectors and Evaluation of Culture Time to Perform Vector Titer by Real-Time Quantitative Polymerase Chain Reaction Assay. Thalass. Rep. 2014, 4, 2189. https://doi.org/10.4081/thal.2014.2189

AMA Style

Baiamonte E, Bagliesi M, Motta V, Spina B, Pecoraro A. Development of Plasmids for Quantitative Detection of Integrated Lentiviral Vectors and Evaluation of Culture Time to Perform Vector Titer by Real-Time Quantitative Polymerase Chain Reaction Assay. Thalassemia Reports. 2014; 4(2):2189. https://doi.org/10.4081/thal.2014.2189

Chicago/Turabian Style

Baiamonte, Elena, Mariella Bagliesi, Valentina Motta, Barbara Spina, and Alice Pecoraro. 2014. "Development of Plasmids for Quantitative Detection of Integrated Lentiviral Vectors and Evaluation of Culture Time to Perform Vector Titer by Real-Time Quantitative Polymerase Chain Reaction Assay" Thalassemia Reports 4, no. 2: 2189. https://doi.org/10.4081/thal.2014.2189

APA Style

Baiamonte, E., Bagliesi, M., Motta, V., Spina, B., & Pecoraro, A. (2014). Development of Plasmids for Quantitative Detection of Integrated Lentiviral Vectors and Evaluation of Culture Time to Perform Vector Titer by Real-Time Quantitative Polymerase Chain Reaction Assay. Thalassemia Reports, 4(2), 2189. https://doi.org/10.4081/thal.2014.2189

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