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

  • Elena Baiamonte UOC Ematologia per le Malattie Rare del Sangue e degli Organi Ematopoietici, Dipartimento di Oncologia ed Ematologia, AO Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy.
  • Mariella Bagliesi UOC Ematologia per le Malattie Rare del Sangue e degli Organi Ematopoietici, Dipartimento di Oncologia ed Ematologia, AO Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy.
  • Valentina Motta UOC Ematologia per le Malattie Rare del Sangue e degli Organi Ematopoietici, Dipartimento di Oncologia ed Ematologia, AO Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy.
  • Barbara Spina UOC Ematologia per le Malattie Rare del Sangue e degli Organi Ematopoietici, Dipartimento di Oncologia ed Ematologia, AO Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy.
  • Alice Pecoraro | a.pecoraro@ospedaliriunitipalermo.it UOC Ematologia per le Malattie Rare del Sangue e degli Organi Ematopoietici, Dipartimento di Oncologia ed Ematologia, AO Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy.

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.

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Published
2014-09-29
Keywords:
vector copy number, real-time quantitative polymerase chain reaction, lentiviral vector titer.
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How to Cite
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). https://doi.org/10.4081/thal.2014.2189