title: Centrifugation facilitates transduction of green fluorescent protein in human monocytes and macrophages by adenovirus at low multiplicity of infection.
abstract: Due to their phagocytic and poorly proliferative nature, it has been difficult to transfect human monocytes and macrophages. Adenoviral vectors have recently allowed transduction of a high percentage of human macrophages, but only after CSF upregulation of the integrins, alphavbeta3 or alphavbeta5, during culture for 48 h, a time allowing significant monocyte to macrophage differentiation. In our hands, after 24-h incubation with M-CSF (20 ng/ml) and a further 24-h incubation with an adenoviral vector encoding green fluorescent protein (AdV-GFP) [multiplicity of infection (MOI)=50:1], only 35% of CD14-positive cells express GFP. We report that centrifugation of these cells with AdV-GFP at 2000 x g for 1 h at 37 degrees C significantly enhanced the number of cells expressing GFP (to 65%) and the level of GFP expression per transduced cell (fivefold). The viability of the cells was not compromised (<5 % CD14-positive cells were 7-aminoactinomycin D (7AAD)-positive after 24 h AdV-GFP exposure at MOI=50:1). Centrifugation allowed efficient transduction of monocytes and macrophages with an MOI at least tenfold lower than otherwise required and did not activate the transduced cells or affect their ability to produce TNFalpha or IL-1beta in response to lipopolysaccharide (LPS). This methodology was also suitable for transducing large numbers of in vitro monocyte-derived macrophages (MDMac) and macrophages isolated from synovial fluids with up to 75-80% of CD14-positive cells transduced after 24-h exposure to AdV-GFP (50:1) and centrifugation (2000 x g). This methodology should provide significant expression of transgenes in human monocytes and macrophages.
authors.affiliation: Department of Microbiology and Infectious Diseases, School of Medicine, Flinders University, GPO Box 2100, Adelaide 5001, Australia.
authors.forename: George C
authors.initials: GC
authors.lastname: Mayne
authors.forename: Romana A
authors.initials: RA
authors.lastname: Borowicz
authors.forename: Kate V L
authors.initials: KV
authors.lastname: Greeneklee
authors.forename: John J
authors.initials: JJ
authors.lastname: Finlay-Jones
authors.forename: Keryn A
authors.initials: KA
authors.lastname: Williams
authors.forename: Prue H
authors.initials: PH
authors.lastname: Hart
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002470:Cell Survival; D002498:Centrifugation / Q000379:methods; D005110:Extracellular Space / Q000737:chemistry; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000096:biosynthesis; D016023:Integrins / Q000096:biosynthesis; D007375:Interleukin-1 / Q000032:analysis; D008164:Luminescent Proteins / Q000235:genetics; D008264:Macrophages / Q000166:cytology*; D009000:Monocytes / Q000166:cytology*; D019030:Receptors, Vitronectin / Q000096:biosynthesis; D013582:Synovial Fluid / Q000166:cytology; D014161:Transduction, Genetic / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
chemical_list: D039302:Integrin alphaVbeta3; D016023:Integrins; D007375:Interleukin-1; D008164:Luminescent Proteins; D019030:Receptors, Vitronectin; D014409:Tumor Necrosis Factor-alpha; C064660:integrin alphaVbeta5; D049452:Green Fluorescent Proteins