Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes.
BACKGROUND
Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection.


RESULTS
Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX) domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes.


CONCLUSIONS
We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.
Johnson
Jennifer L
JL
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, USA. jjohnson@scripps.edu <jjohnson@scripps.edu>
Ellis
Beverly A
BA
Munafo
Daniela B
DB
Brzezinska
Agnieszka A
AA
Catz
Sergio D
SD
D002454:Cell Differentiation / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D018922:HL-60 Cells; D006801:Humans; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000235:genetics*; D009504:Neutrophils / Q000378:metabolism*; D010588:Phagosomes / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics
D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1