According to the Human Genome Project website, there are approximately 20,000 protein-coding
genes within the three billion or so chemical base pairs that make up human DNA.
Identifying the genes, however, was just a first step in understanding humans at
the molecular level. For most of those 20,000 genes, their function is still
an open question.
Millions of dollars have been devoted to developing methods to precisely control
specific genes – to eliminate or alter them so that their protein products
aren’t produced -- in order to learn how an organism differs when the gene
is functioning properly and when it isn’t.
In the last 30 years, scientists have developed the ability to “knock out”
genes in model organisms like yeast, fruit flies, zebra fish, and mice by replacing
or disrupting the “gene of interest” with an artificial piece of DNA.
The development of “knockout” organisms has greatly benefitted medical
research by enabling researchers to observe links between genotypic change (gene
mutation) and phenotypic (physical) changes. Developing a line of knockout
mice is a costly and lengthy process, however, and the techniques are not effective
for all types of cells.
In the last decade, scientists discovered a low cost and fast way to silence genes
of interest by using a cell’s own mechanisms to inhibit a gene’s function
-- keeping the gene intact, but stopping it from making its protein product.
The technology, called RNA interference (RNAi) is effective even in types of cells
that are difficult or impossible to genetically alter by other methods.
to a grant from the Children’s Discovery Institute and the leadership of Dr.
Sheila Stewart (left), assistant professor of cell biology and physiology at Washington
University School of Medicine, and her collaborators, this technology has been made
available at very low cost to Institute investigators.
RNAi gene silencing targets a key molecule that helps turn the genetic information
encoded in DNA into proteins – messenger RNA.
Messenger RNA (mRNA) is formed in the nucleus of a cell from a DNA gene template.
This is called transcription. In mRNA as in DNA, specific sequences of nucleotides
encode the genetic information. While DNA never leaves the nucleus, it is the mRNA
that carries the encoded chemical recipe dictated by the gene from the nucleus to
sites within the cell where proteins are created.
“Attacking the messenger” in the case of genetic research provides an
important tool for understanding protein function. Without the recipe carried
by mRNA, the protein specified by the gene cannot be produced. Destroy the
mRNA and you do away with the protein.
The technology is based upon a discovery made in the late 1980s of a mechanism cells
use to defend against infection by certain viruses that have double stranded RNA.
Simply put, when a virus infects a cell by putting its double stranded RNA into
a cell, the cell mounts a defense that cuts apart and degrades the invading RNA.
The scientists who first discovered this mechanism, Andrew Fire and Craig Mello,
received the 2006 Nobel Prize in Medicine.
Other scientists figured out ways to fool the cell into seeking out and destroying
specific messenger RNAs that originated in its own nucleus along with the foreign
RNA. If the nucleotide sequence of an RNA provided by the researcher matches
the sequence of the mRNA to be destroyed, the cell’s molecular defenses seek
Today, researchers create tiny tailor made snippets of RNA with nucleotide sequences
patterned after the sequences of the specific genes they wish to silence.
This so called “silencing RNA” can be packaged in viruses, and the viruses
can be used to “infect” a cell.
Once the double stranded RNA is inside the cell, a special enzyme called Dicer chops
up the silencing RNA into small bits of no more than 50 nucleotides in length.
These small bits are combined into a complex called the RNA-induced silencing complex
(RISC), which targets and degrades the “look alike” mRNA in the cell.
If all goes well, the cell responds as if the gene never existed, giving researchers
clues about its function.
Dr. Stewart played a key role in making RNAi a powerful tool in biological research.
As a postdoctoral fellow at the Whitehead Institute for Biomedical Research at MIT
between 1998 and 2003, she designed the viral vector that delivers the synthesized
RNA into the cell.
The Whitehead Institute was one of founding members of a collaboration of academic
research institutions and life science organizations that joined together in the
early 2000s as “The RNAi Consortium.”
This Boston-based consortium has built a library of 160,000 custom-designed RNAi
constructs targeting 15,000 human genes and 15,000 mouse genes. They also developed
methods to apply this library effectively for loss-of-function genetic screens,
enabling investigators to examine gene function in cell lines, primary cultures,
and animals to identify novel genes that play critical roles in human disease.
Like most new breakthrough technologies, RNAi doesn’t come cheap. Production
costs to develop the specific RNA inhibitors have exceeded $18 million. The
price of a library copy (which is frozen copies of the various RNA constructs) exceeds
Because of her key contributions to the Consortium, Dr. Stewart, who joined Washington
University School of Medicine in 2003, was able to bring a copy of the Consortium’s
library to Washington University.
For the library to be a useful tool, however, Stewart needed infrastructure to make
copies of the library plates, develop DNA stocks of high enough quality to transfect
cells, and do viral production and verification. “To go from library stocks
to a reliable tool for gene silencing still requires many costly steps,” says
Stewart. “That’s what the Discovery Institute grant has enabled.”
Funding from the Discovery Institute - along with assistance from Washington University’s
Genome Sequencing Center and the University’s High Throughput Core - has given
Discovery Institute members RNAi gene control at a fraction of the cost charged
by outside suppliers, a cost that will allow the library to be sustained for the
Dr. Stewart’s collaborators in the RNAi Core include Discovery Institute member
Dr. Elaine Mardis who co-directs the Genome Sequencing Center at Washington University,
and Dr. David Piwnica-Worms, Professor of Radiology and Developmental Biology at
Washington University School of Medicine.