title: Novel centrifugal method for simple and highly efficient adenovirus-mediated green fluorescence protein gene transduction into human monocyte-derived dendritic cells.
abstract: Dendritic cells (DC) are professional antigen-presenting cells in the immune system. Gene transduction of DC with tumor-associated antigen (TAA) or other genes that enhance the immune reaction has been considered theoretically useful for DC-based immunotherapy. However, gene transduction of DC generated from human peripheral blood monocytes has been difficult due to its low efficiency, even when adenoviral vector was used at high multiplicity of infection (MOI). In the present study, we examined the effect of centrifugal force to enhance efficiency of adenovirus-mediated gene transduction into human monocyte-derived DC at various rotor speeds at various temperatures for various times. We judged the transduction efficiency using enhanced green fluorescence protein (EGFP)-expressing adenoviral vector, and the best condition for centrifugal transduction was determined as 2000 x g at 37 degrees C for 2 h at an MOI of 10 or greater. At an MOI of 50 without centrifugation, the gene transduction efficiency was about 66% and mean fluorescence intensity (MFI) of EGFP expression was about 150 (at 37 degrees C for 2 h). With centrifugal transduction (2000 x g at an MOI of 50 at 37 degrees C for 2 h), 86% or more DC were gene-modified, and especially, MFI of EGFP expression was highly enhanced (MFI: about 3100 or greater). Centrifugally gene-transduced DC were not damaged and were thoroughly functional as measured by mixed lymphocyte reaction (MLR). The centrifugal method was also applicable to human monocytes and K562 cells. The centrifugal transduction method with adenoviral vector might be helpful for the generation of gene-modified DC.
authors.affiliation: Third Department of Internal Medicine, School of Medicine, University of Tokushima, Kuramoto-cho 3, 770-8503, Tokushima, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Nishimura
authors.forename: Y
authors.initials: Y
authors.lastname: Nishioka
authors.forename: T
authors.initials: T
authors.lastname: Shinohara
authors.forename: H
authors.initials: H
authors.lastname: Ogawa
authors.forename: S
authors.initials: S
authors.lastname: Yamamoto
authors.forename: K
authors.initials: K
authors.lastname: Tani
authors.forename: S
authors.initials: S
authors.lastname: Sone
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D017951:Antigen Presentation; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D002498:Centrifugation / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D003713:Dendritic Cells / Q000276:immunology*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D020014:K562 Cells; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D009000:Monocytes / Q000502:physiology*; D010641:Phenotype; D013696:Temperature; D013997:Time Factors; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection
chemical_list: D004279:DNA, Viral; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins