title: Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors.
abstract: BACKGROUND
  Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages.
  
  
  RESULTS
  Human blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494.
  
  
  CONCLUSIONS
  This report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages.
authors.affiliation: Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Leyva
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Anzinger
authors.forename: J Philip
authors.initials: JP
authors.lastname: McCoy
authors.forename: Howard S
authors.initials: HS
authors.lastname: Kruth
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D005434:Flow Cytometry; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016173:Macrophage Colony-Stimulating Factor