National Heart, Lung, and Blood Institute Division of Intramural Research
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Fly Embryo RNAi Project
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The RNA-mediated interference (RNAi) has been reported to be a simple and powerful method to down-regulate endogenous gene function in a number of organisms including planaria, Caenorhabditis elegans, Drosophila, and mice. Injection of dsRNA corresponding to a specific gene into organisms silences expression of the specific gene by rapid degradation of the corresponding mRNA in affected cells.

The Mechanism of RNAi

RNAi involves at least two important steps: the initiation step and the effector step. In the initiation step in Drosophila, a type III RNase, "Dicer", processes long dsRNAs into double-stranded, small interfering RNAs (siRNA), which are 21-23 nucleotides (nt) long. Effector step starts once siRNAs are produced. They trigger the formation of RNA-induced silencing complexes (RISC), the protein-RNA effector nuclease complxes. A helicase in the complex unwinds the siRNA, resulting in single-stranded RNA (ssRNA), which is used as a guide for substrate selection. Once the ssRNA is base-paired with the target mRNA, the mRNA is destroyed by the RISC (view a diagram showing the proposed RNA degradation process).

Embryonic Development Studies

At National Heart, Lung, and Blood Institute, RNAi has been used as a reverse genetic tool to study genes involved in Drosophila embryonic development. Headed by Dr. Marshall Nirenberg, chief of the Laboratory of Biochemical Genetics, Andrey Ivanov, Alessandra Rovescalli, Paola Pozzi, Brian Mozer, Hsi-Ping Li, Siuk Yoo, Haruhiro Higashida, Shu-hua Yu, and Vicky Guo have been focussing on central and peripheral nervous systems. Drs. Yongsok Kim (chief of RNAi Core Facility), Yong-Ou Kim, and Sang-Joon Park have been screening genes involved in heart development.

A String with D-mef2-lacZ Transgene

The yw strain carrying D-mef2-lacZ transgene has been used as the source of embryos for heart development research. This gene is a beta-galactosidase (ß-gal) marker gene that is expressed in the cardiac cells and a subset of ventral muscle founder cells. Because the ß-gal marker gene is expressed in cardiac cells throughout heart development, simple X-gal staining followed by monitoring of the ß-gal expression pattern and heart morphology in injected embryos enabled us to successfully identify potential cardiogenic genes.

Screen Heart Genes

A genome-wide search has been performed for cardiogenic genes that are essential for development of the Drosophila embryonic heart. For the screen, dsRNAs were microinjected into host Drosophila embryos. As a dsRNA source, master plates were prepared. The master plates (96-well plates) contained a total of 5849 individual long dsRNAs, representing all the genes of the Drosophila Gene Collection (DGC 1.0) released by the Berkely Drosophila Genome Project. In addition, we also synthesized short dsRNAs (21-mer) for a group of genes that were not included in DGC 1.0. I Initially, embryos (40-60 embryos per pool) were injected with a pool of dsRNAs of three different genes. From the X-gal staining patterns in the injected embryos, a positive pool was identified, and then the individual dsRNA from a positive pool was injected again to determine which gene was silenced and thereby caused the phenotype. Because scientists often further characterized injected embryos by immunostaining with specific antibodies, a total of several hundred embryos were injected with dsRNAs to identify one specific gene that causes a reproducible mutant heart phenotype (Kim et al., 2004).

Monoclonal Antiboy 22C20

To visually identify potential genes controlling nervous system development, antibody 22C10 has been widely used. This antibody recognizes specific epitopes present in the cytoplasm and the inner surface of cell membranes of all PNS (peripheral nervous system) neurons and a subset of CNS(central nervous system) neurons. DAB (diaminobenzidine) staining has been used for visualization under a brightfield microscope, and fluorescent secondary antibody staining has been used for confocal and Two-Photon microscope observations.

Screen Nervous System Genes

5,800 dsRNAs were synthesized from cDNA-plasmid unigene set I (Berkeley) by in vitro transcription. dsRNAs were microinjected into pre-blastoderm embryos and the phenotype was determined by antibody staining specific for the central and peripheral nervous systems. More and more genes that affect neural development have been identified. Interestingly, some genes displayed novel RNAi phenotypes. These include SAK (Snk/Plk-Akin Kinase), Casein Kinase II beta, Bx-42, and PAG (Phospho-Acetyl Glucosamine mutase).

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Fly Embryo RNAi Project (Version 2.10)