title: Protein overexpression following lentiviral infection of primary mature neutrophils is due to pseudotransduction.
abstract: Neutrophils are terminally differentiated cells with a short life-span due to constitutive apoptosis. Because of these characteristics, genetic manipulation of neutrophils has been difficult, although it is highly desired given the importance of neutrophils in the immune system. Here we demonstrate that transduction of primary human mature neutrophils with enhanced green fluorescent protein (eGFP)-encoding lentiviral particles results in GFP-containing cells as previously reported. Yet, our data further show that GFP expression in neutrophils upon transduction is largely due to protein transfer, a process called lentiviral pseudotransduction, and not due to bona fide transduction. Thus, inhibition of viral genome integration by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) or of protein biosynthesis by cycloheximide (CHX) did not abolish GFP levels in transduced neutrophils. Importantly, lentiviral pseudotransduction of the enzyme death-associated protein kinase 2 (DAPK2) into primary human mature neutrophils resulted in increased protein levels, but not enzymatic functionality. Based on our data and previous reports of unspecific viral effects on immune cells following lentiviral transduction, we discourage scientists to use lentiviral transduction methods to manipulate primary mature neutrophils.
authors.affiliation: Institute of Pharmacology, University of Bern, Bern, Switzerland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Geering
authors.forename: Jan
authors.initials: J
authors.lastname: Schmidt-Mende
authors.forename: Elena
authors.initials: E
authors.lastname: Federzoni
authors.forename: Christina
authors.initials: C
authors.lastname: Stoeckle
authors.forename: Hans-Uwe
authors.initials: HU
authors.lastname: Simon
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D064550:Death-Associated Protein Kinases; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D016086:Lentivirus / Q000235:genetics*; D009154:Mutation; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D018894:Reverse Transcriptase Inhibitors; D049452:Green Fluorescent Proteins; D015215:Zidovudine; D003513:Cycloheximide; C579104:DAPK2 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases