Stable Gene Silencing in Human Monocytic Cell Lines Using Lentiviral-delivered Small Interference RNA

Abstract

Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. To overcome this problem, we developed a system for mediating RNA interference in monocytic cells. The p110α isoform of phosphoinositide 3-kinases (PI3Ks) was silenced using a lentiviral vector expressing short hairpin RNA (shRNA). This resulted in the generation of stable THP-1 and U-937 monocytic cell lines deficient in p110α. Notably, p110α was silenced without affecting levels of either the other class IA PI3K catalytic subunits p110β and p110δ, or the p85α regulatory subunit. The role of p110α in mediating cell adherence was examined. Monocyte adherence induced in response to either lipopolysaccharide (LPS) or 1α,25-dihydroxycholecalciferol (D3) was blocked by the PI3K inhibitor LY294002. However, although adherence induced in response to D 3 was sensitive to silencing of p110α, LPS-induced adherence was not. Expression of the monocyte differentiation marker CD11b was also induced by D3 in a PI3K-dependent manner and gene silencing using shRNA showed that p110α was also required for this effect. Taken together, these findings demonstrate that LPS and D3 use distinct isoforms of class IA PI3K to induce functional responses and that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology.