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.
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).
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
Hsi-Ping Li, Siuk Yoo, Haruhiro
Higashida, Shu-hua Yu, and Vicky
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.
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
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).
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.
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).