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 ZnSO(4), hence allowing for virus titration by transducing HeLa cells with serially diluted virus, subsequent ZnSO(4) 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.
authors: Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
authors: Wen-Hsin
authors: WH
authors: Lo
authors: Chi-Yuan
authors: CY
authors: Chen
authors: Chia-Ni
authors: CN
authors: Yeh
authors: Chin-Yu
authors: CY
authors: Lin
authors: Yu-Chen
authors: YC
authors: Hu
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism / Q000821:virology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism / Q000821:virology; D006801:Humans; D011401:Promoter Regions, Genetic; D011817:Rabbits; D013997:Time Factors; D014161:Transduction, Genetic / Q000379:methods*; D019287:Zinc Sulfate / Q000494:pharmacology
chemical_list: D055396:Bone Morphogenetic Protein 2; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019287:Zinc Sulfate