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Rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
17568 42680 2011 6 PDF Available
Title
Rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells
Abstract

Baculovirus is a promising gene delivery vector and can be titrated by constitutive EGFP expression in HeLa cells, which, however, might interfere with target transgene expression and impart cytotoxicity. Here we constructed Bac-ME accommodating egfp under the inducible metallothionein promoter and Bac-MECB harboring an additional BMP-2 gene. Bac-ME effectively transduced HeLa cells with minimal leaky expression, but expressed EGFP robustly upon induction with ZnSO4, hence allowing for virus titration by transducing HeLa cells with serially diluted virus, subsequent ZnSO4 induction and flow cytometry analysis of EGFP-positive cells. The titration protocol enabled the generation of discernable titration curves, determination of transducing titers, and discrimination of the transducing abilities of different virus batches. After titration, cell transduction with pre-determined Bac-ME dose revealed consistent transduction efficiency dependence on the dose, regardless of virus batch and cell type. Bac-MECB was similarly titrated by inducible EGFP expression and used to transduce de-differentiated articular chondrocytes without EGFP induction. BMP-2 expression was proportional to the Bac-MECB dose and promoted cartilage-specific matrix synthesis, implicating the potential of Bac-MECB in restoring chondrocyte differentiation. These data confirmed that regulatable EGFP expression enabled rapid, reliable baculovirus titration without interference with subsequent applications.

Keywords
Baculovirus; EGFP; Inducible promoter; Metallothionein; Titration; Transducing titer
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Rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 48, Issue 1, 5 January 2011, Pages 13–18
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us