Systems Biological Study of Drosophila Immune Signaling
Acta Universitatis Tamperensis, No. 1599
By Jenni Kallio
Tampere University Press
Distributed by Coronet Books
$82.50 Paper Original
The development of systems biological high-throughput methods such as microarray analysis and genome-wide screens based on ribonucleic acid interference (RNAi) opened many doors for scientific research. Vast amounts of data can be obtained from a biological system and processed, while the system functions. . The ambitious goal of systems biological studies is to characterize the entire biological system: to identify all of its components and to determine how the system is regulated. However, a large scale systems biological study requires careful planning prior to experiments and taking into account any problems that might be related to the assay.
In this study, we used two systems biological methods, an oligonucleotide-microarray assay and a genome-wide RNAi screen, to characterize the Drosophila melanogaster immune response. Drosophila provides an excellent model for studying the principles of innate immunity. The fruit fly’s evolutionary conserved immune system lacks adaptive immunity, so it relies solely on its innate immunity for defending against pathogens. In addition, many genetic and molecular techniques, including in vivo RNAi, are available for Drosophila enabling effective exploitation of the data obtained from in vitro studies. Also, Drosophila are cheap and easy to maintain, and RNAi in Drosophila is effective and straightforward to carry out.
In our systems biological study of the Drosophila innate immunity, we first used a microarray analysis to identify new gene products involved in innate immune signaling and phagocytosis. A microarray analysis was carried out for the genes induced in response to E. coli in Drosophila S2 cells. This was followed by an RNAi-based functional analysis of the up-regulated genes. Second, we carried out two separate genome-wide RNAi in vitro screens to identify gene products necessary for Drosophila immune signaling: the Drosophila nuclear factor kappa B (NF-?B) signaling and the Drosophila Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. We identified ten novel regulators of the Drosophila NF-?B signaling and five novel regulators of the Drosophila JAK/STAT pathway.
One gene from both RNAi screens was subjected to further functional studies. We carried out the in vitro characterization of G protein-coupled receptor kinase 2 (Gprk2) identified in the NF-?B screen. Similarly, we also characterized Eye transformer (ET), which was identified in the JAK/STAT screen. We were able to confirm both original phenotypes with targeted RNAi treatments. Furthermore, we validated these results by quantitative reverse transcription polymerase chain reaction (RT-PCR). Thereafter, we characterized both genes in vivo.
In our Drosophila in vivo RNAi assays, transgenic upstream activating sequence (UAS)-RNAi flies were crossed with selected GAL4 driver flies, generating RNAi silencing of the target gene in the offspring. These flies were infected with E. cloacae to induce immune signaling and the RNAi phenotypes were validated by qRT-PCR from RNAs extracted from the flies. We concluded that Gprk2 has an essential role in the Drosophila Toll pathway mediated immunity in vivo, and that ET is a negative regulator of Drosophila Tot gene expression in vivo.
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