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Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells
Author(s) -
Böcker Wolfgang,
Rossmann Oliver,
Docheva Denitsa,
Malterer Georg,
Mutschler Wolf,
Schieker Matthias
Publication year - 2007
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1049
Subject(s) - ex vivo , transduction (biophysics) , biology , transgene , microbiology and biotechnology , viral vector , titer , stem cell , mesenchymal stem cell , lentivirus , cell culture , genetic enhancement , virology , in vivo , virus , gene , recombinant dna , genetics , biochemistry , viral disease
Background Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long‐term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time‐consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. Methods Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. Results Third‐generation self‐inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. Conclusions Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs. Copyright © 2007 John Wiley & Sons, Ltd.