Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA

Silencing of the p110α isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1α,25-dihydroxycholecalciferol and bacterial lipopolysaccharide

Jimmy S. Lee, Zakaria Hmama, Alice Mui, Neil E. Reiner

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41 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)9379-9388
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number10
DOIs
StatePublished - Mar 5 2004
Externally publishedYes

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1-Phosphatidylinositol 4-Kinase
Calcitriol
Gene Silencing
Phosphatidylinositols
RNA Interference
Lipopolysaccharides
Protein Isoforms
Phosphotransferases
Genes
Cells
RNA
Cell Line
Small Interfering RNA
Transfection
Monocytes
Cytology
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Differentiation Antigens
Phagocytes
Cell Biology

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA: Silencing of the p110α isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1α,25-dihydroxycholecalciferol and bacterial lipopolysaccharide",
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.",
author = "Lee, {Jimmy S.} and Zakaria Hmama and Alice Mui and Reiner, {Neil E.}",
year = "2004",
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T1 - Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA

T2 - Silencing of the p110α isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1α,25-dihydroxycholecalciferol and bacterial lipopolysaccharide

AU - Lee, Jimmy S.

AU - Hmama, Zakaria

AU - Mui, Alice

AU - Reiner, Neil E.

PY - 2004/3/5

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N2 - 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.

AB - 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.

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