Different roles of G protein subunits β1 and β2 in neutrophil function revealed by gene expression silencing in primary mouse neutrophils

Abstract

Neutrophils play important roles in host innate immunity and various inflammation-related diseases. In addition, neutrophils represent an excellent system for studying directional cell migration. However, neutrophils are terminally differentiated cells that are short lived and refractory to transfection; thus, they are not amenable for existing gene silencing techniques. Here we describe the development of a method to silence gene expression efficiently in primary mouse neutrophils. A mouse stem cell virus-based retroviral vector was modified to express short hairpin RNAs and fluorescent marker protein at high levels in hematopoietic cells and used to infect mouse bone marrow cells prior to reconstitution of the hematopoietic system in lethally irradiated mice. This method was used successfully to silence the expression of Gβ1 and/or Gβ2 in mouse neutrophils. Knockdown of Gβ2 appeared to affect primarily the directionality of neutrophil chemotaxis rather than motility, whereas knockdown of Gβ1 had no significant effect. However, knockdown of both Gβ1 and Gβ2 led to significant reduction in motility and responsiveness. In addition, knockdown of Gβ1 but not Gβ2 inhibited the ability of neutrophils to kill ingested bacteria, and only double knockdown resulted in significant reduction in bacterial phagocytosis. Therefore, we have developed a short hairpin RNA-based method to effectively silence gene expression in mouse neutrophils for the first time, which allowed us to uncover divergent roles of Gβ1 and Gβ2 in the regulation of neutrophil functions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.