Comparison of lentiviral vector titration methods - PubMed (original) (raw)

Comparative Study

Comparison of lentiviral vector titration methods

Martine Geraerts et al. BMC Biotechnol. 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.

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Figures

Figure 1

Figure 1

Overview of primer sets and lentiviral vector constructs. (A) Schematic representation of CH-eGFP-WS lentiviral vector with the corresponding amplicons of the different primer sets (LTR-gag, GFP and WPRE). Primer sequences are represented in small caps and probe sequences are in bold. (B) Schematic representation of different lentiviral vector constructs. The construct was optimized to increase transduction efficiency (cPPT and WPRE) and biosafety (SIN) as described before [9, 10, 23].

Figure 2

Figure 2

Comparison of lentiviral vector titration methods. CH-eGFP-WS vector was serially diluted (1/2) and subjected to RT-qPCR, ELISA and FACS analysis after transduction of 293T cells, to determine the linearity of the different titration methods. The correlation coefficients are representative for 3 independent experiments. The asterix (*) represents values that were not included in the linear regression.

Figure 3

Figure 3

Kinetics of lentiviral vector production. The CH-eGFP-WS-derived lentiviral vector was produced in two CF2 modules by triple transient transfection in serum-free medium. Starting at 2 days post-transfection, cell-culture medium was harvested once a day for five consecutive days and concentrated by low-speed centrifugation. The p24 content (pg p24/ml) of vector preparations was determined by ELISA, the transducing titers (TU/ml) by FACS analysis and the RNA equivalents (RNA/ml) by one-step RT-qPCR with LTR primers. Data represent the mean value ± standard deviation and represent 3 independent experiments. A decrease in vector titer starting at 3 days post-transfection was evidenced by all methods. The specific activities (TU/pg and TU/RNA) are depicted in the inset.

Figure 4

Figure 4

Correlation of eGFP fluorescence and mRNA transgene expression levels. Four independently produced preparations of CH-eGFP-WS-derived lentiviral vector were serially diluted (1/10) prior to transduction of 293T cells. 6 days later, cells were harvested for eGFP analysis by FACS or RNA extraction and subsequent RT-qPCR with primers and probe directed against WPRE to measure transgene expression and against RNAse P to normalize for total RNA content. Total eGFP expression was measured by multiplying the percentage of transduced cells (TE) with the mean fluorescence intensity (MFI). The transgene mRNA level is given as the number of mRNA copies normalized to the total RNA content (RNAc/ng). These values correlate strongly with each other (r2 = 0.97).

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