Determination of the baculovirus transducing titer in mammalian cells

Abstract

Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments.

Adaptive Immune Responses Elicited by Baculovirus and Impacts on Subsequent Transgene Expression In Vivo

“…Bac-CE expressing enhanced green fluorescent protein (EGFP) as the reporter was constructed as described previously (30). To construct pBac-luc/w, the firefly luciferase gene was amplified from pGEM-luc (Promega) and cloned into pBac-CMV5 (whose polyhedrin promoter was replaced by the cytomegalovirus immediate-early [CMV-IE] promoter [31]).…”

“…To measure the transducing titer, sera were diluted 20-fold with PBS and mixed with an equal volume of Bac-CE (5 ϫ 10 7 PFU in 100 l) at 4°C for 2 h. HeLa cells were transduced with the mixture containing the neutralized Bac-CE and cultured for 24 h. The transducing titer was calculated as described previously (30).…”

“…To quantify the effect of neutralizing antibodies on BV transduction, the transducing titer of Bac-CE, after mixing with serum, was measured as described previously (30) and is expressed as transducing units (TU)/ml (Fig. 2D).…”

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle

“…The viral titer was determined based on the ability of virus to transduce mammalian cells using a recently developed protocol [19] and was 3 × 10 7 transducing units per milliliter (TU/ml). The virus dosage per cell (TU/cell) is expressed as multiplicity of transduction (MOT).…”

Concanavalin a affinity chromatography for efficient baculovirus purification

“…The transducing titers were defined as transducing units per milliliter (TU/mL) and were measured based on the ability of baculovirus to transduce HeLa cells after serial dilution and titration. 37,38 The VP were quantified by a flow cytometry-based method. 38,39 Briefly, baculovirus was first diluted with DPBS to %10 6 particles/mL (%10 2 to 10 4 dilution).…”