Factors influencing the production and storage of baculovirus for gene delivery: An alternative perspective from the transducing titer assay

Tsai, Chien-Tai; Chan, Zun-Ren; Lu, Juin-Ming; Yang, Ding-Gang; Lo, Wen-Hsin; Hu, Yu‐Chen

doi:10.1016/j.enzmictec.2006.10.006

Cited by 10 publications

(11 citation statements)

References 33 publications

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“…Cultures were allowed to grow for 18–24 hours to a viable cell density of ~1.5 × 10 6 cells/mL for infection at a multiplicity of infection (MOI) of 0.1 pfu/cell using a virus stock of ~5 × 10 7 pfu/mL. This MOI is commonly used for virus amplification (Tsai et al, 2007). After infection, the culture was incubated at 27°C on a stir plate set at 100 rpm.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the Virus Counter® for rapid baculovirus quantitation

Ferris¹,

Stepp²,

Ranno³

et al. 2011

Journal of Virological Methods

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The utility of a new instrument for rapid virus quantitation, the Virus Counter, was evaluated in a blind study conducted at three sites. This instrument is a substantially improved version of the original academic research instrument described previously by Stoffel et al. (2005a). The addition of hydrodynamic focusing, a self-contained fluidics system and customized software for system control and data analysis has resulted in a commercially viable and available design.

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Section: Methodsmentioning

confidence: 99%

Evaluation of the Virus Counter® for rapid baculovirus quantitation

Ferris¹,

Stepp²,

Ranno³

et al. 2011

Journal of Virological Methods

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“…This protocol mimics the titration of retroviral or AAV vector by serially diluting the EGFP-expressing virus, followed by transduction in the multi-well plates and flow cytometry analysis of baculovirus-mediated EGFP expression, and therefore directly measures the biological activity of baculovirus. This method enables rapid and reliable titration of baculovirus and is helpful in predicting the outcome of gene delivery experiments [37] and monitoring the baculovirus production [140] and purification (unpublished data) processes.…”

Section: Techniques For Baculovirus Quantification and Purificationmentioning

confidence: 98%

Baculoviral Vectors for Gene Delivery: A Review

2008

CGT

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131

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Baculovirus has emerged as a novel vector for in vivo and in vitro gene delivery. In addition, the applications of baculovirus-mediated gene transfer have been explosively expanded to drug screening, eucaryotic gene display, cancer therapy and tissue engineering, etc. The capability of baculovirus viral envelope for protein/peptide display also renders itself a potential vaccine delivery platform. This paper reviews the history, factors influencing baculovirus-mediated gene delivery and emerging in vitro, in vivo and ex vivo applications. Efforts aimed at overcoming current existing bottlenecks and recent progresses in addressing the safety concerns are particularly emphasized.

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“…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).…”

Section: Quantification Of Transducing Titers and Viral Particlesmentioning

confidence: 99%

“…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). To fix the viral particles, 950 lL diluted virus was mixed with 20 lL formaldehyde (5%) and incubated at 4 C for 45 min.…”

Section: Quantification Of Transducing Titers and Viral Particlesmentioning

confidence: 99%

Concanavalin a affinity chromatography for efficient baculovirus purification

Chen¹,

Chen²,

Chang³

et al. 2009

Biotechnology Progress

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Baculovirus has emerged as a novel gene delivery and vaccine vector, and the demand for purified baculovirus is rising due to the increasing in vivo applications. Since the baculoviral envelope protein gp64 is a glycoprotein, we aimed to develop a concanavalin A (Con A) chromatography process, which harnessed the possible affinity interaction between gp64 and Con A, for simple and effective baculovirus purification. Throughout the purification process the virus stability and recovery were assessed by quantifying the virus transducing titers [TT, defined as transducing units (TU) per milliliter] and viral particles (VP). We found that baculovirus stability was sensitive to buffer conditions and diafiltration with a tangential flow filtration system LabScale using 300 K membranes yielded recoveries of approximately 75% in TT and 82% in VP. The diafiltered baculovirus strongly bound to the Con A column as evidenced by the low virus losses to the flow through and wash fractions. The wash steps eliminated >99% of protein impurities and elution with 0.6 M alpha-D-methylmannoside at room temperature led to the recoveries of approximately 16% in VP and approximately 15.3% in TU. The resultant VP/TU ratio was as low as 41.4, attesting the high quality of the purified virus. Further elution with 1 M alpha-D-methylmannoside recovered another 6% virus TU, yielding a cumulative recovery of approximately 21.3% in TU. These data demonstrated for the first time that Con A chromatography is suitable for baculovirus purification, and may be used for the purification of other viruses with surface glycoproteins.

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Factors influencing the production and storage of baculovirus for gene delivery: An alternative perspective from the transducing titer assay

Cited by 10 publications

References 33 publications

Evaluation of the Virus Counter® for rapid baculovirus quantitation

Evaluation of the Virus Counter® for rapid baculovirus quantitation

Baculoviral Vectors for Gene Delivery: A Review

Concanavalin a affinity chromatography for efficient baculovirus purification

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