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Chapter 35 : Whole Genome Screens in Macrophages

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Whole Genome Screens in Macrophages, Page 1 of 2

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Abstract:

The development of whole genome RNA interference (RNAi) by using double-stranded RNA (dsRNA) in cells opened up the potential to screen the entire genome of macrophage-like cells for host proteins that modulate bacterial infection. This chapter focuses on how these screens have been used in in vitro studies with several different pathogens, both intra- and extracellular. The S2 cell line is thought to be derived from embryonic plasmatocytes and behaves similarly to primary macrophages. Coupled with the fact that lack the interferon response, allowing use of (relatively) inexpensive dsRNAs (unlike mammalian cells), and that S2 cells take up dsRNAs passively through the receptor Eater, it is reasonable that whole genome screens for macrophage function using RNAi first became viable in cell lines. A remarkably small group of genes appear to be required for phagocytosis of all the pathogens studied so far, and most of these are concerned with actin remodeling and vesicle trafficking. The power and usefulness of genome-wide screens utilizing RNAi technology depends on the critical assumption that gene silencing is a very specific process.

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35

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FIGURE 1

Schematic outline of different phenotypes in fluorescent-based macrophage-pathogen screens. (A) ( ): (i) normal phagocytosis by S2 cells; (ii) decreased phagocytosis after RNAi; (iii) “spot” phenotype (see text); (iv) increased bacterial viability within S2 cell. (B) ( ). The screen utilized GFP-expressing . Nonphagocytosed cells were distinguished from phagocytosed yeast by secondary staining (thick outline) with an anti- antibody. (C) ( ). This screen utilized GFP under a macrophage-activated promoter in the bacteria (i). Non-fluorescence could signify nonphagocytosed cells (ii) or phagosome-vacuolar escape (iii).

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35
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Image of FIGURE 2
FIGURE 2

Schematic showing pathogen-specific factors in S2 cells identified by RNAi and other screens.

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35
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Tables

Generic image for table
TABLE 1

Genes implicated in phagocytosis by at least three independent screens

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35
Generic image for table
TABLE 2

Summary of genome-wide phagocytosis screens

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35

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