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Open Access Highly Accessed Research article

Comparison of lentiviral vector titration methods

Martine Geraerts1, Sofie Willems1, Veerle Baekelandt2, Zeger Debyser1* and Rik Gijsbers1

Author Affiliations

1 Laboratory for Molecular Virology and Gene Therapy, K.U.Leuven and IRC KULAK, Flanders, Belgium

2 Laboratory for Neurobiology and Gene Therapy, K.U.Leuven, Flanders, Belgium

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BMC Biotechnology 2006, 6:34  doi:10.1186/1472-6750-6-34

Published: 12 July 2006

Abstract

Background

Lentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression.

Results

We compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes.

Conclusion

The different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others.