Boston, MA -- April 16, 2002 -- In a study released in the April 16 Proceedings of the National Academy of Sciences, Harvard Medical School researchers report a new technique that can "silence" or knock-down the production of specific proteins in mammalian cells. The method has potential as a tool to speed the investigation of protein function--a field opened up by the sequencing of the human and other genomes. And it may eventually be used as a platform for developing new therapies.
The availability of the new method--vector-based RNA interference--extends the range of possibilities of selective interference of gene expression without having to manipulate DNA in the egg or embryo. The vector-based technology makes it possible to turn off genes in a highly specific manner in the mature cell, and its effect is persistent unlike earlier RNA interference methods for human cells. Previously, to study mammalian protein function, many researchers had to go the laborious route of making knockout animals, a process that can take months. The new technology enables them to complement this technology and conduct a variety of experiments with cells grown in vitro, many of which represent differentiated cell types often difficult to obtain as primary cultures.
The technology was developed by researchers in the laboratory of Yang Shi, associate professor of pathology at the Medical School. They constructed a special plasmid vector--a ring of DNA that carries foreign genetic material into cells. Once inside, the vector directs the enzyme RNA polymerase to make snippets of double-stranded RNA. These then bind to matching strands of messenger RNA, neutralizing it and causing it to be degraded, thus interfering with protein synthesis.
This effect of "knocking down" a gene's expression has broad practical applications--for example, to screen for protein function by seeing what happens to an organism when the protein is absent. This is the goal of the growing fields of proteomics and genomics, which try to determine the function of proteins identified by whole genome sequencing.
"I'm sure everyone is going to try these experiments now the DNA vector has been shown to work," said Shi.
The method Shi and coworkers used, RNA interference, had already proven its general utility in other non-mammalian systems, like the fruit fly, and in plants, fungi, and the worm C. elegans. In these organisms, double-stranded RNA injected into cells inhibits the synthesis of protein made by the gene that matches the injected RNA. But parallel experiments failed in mammalian cells, depriving researchers of a valuable tool for studying mammalian protein function.
"For a long while people were so jealous of C. elegans researchers because they could do RNA interference," said Shi.
Then, in what Shi called a "groundbreaking" discovery last year, researchers in Germany inserted very short--21 nucleotide--snippets of RNA into mammalian cells grown in tissue culture. These smaller, double-stranded RNA fragments were able to evade the protective response the cell made to longer double-stranded RNA, the cause of earlier failures.
The vector system devised by Shi and coworkers represents the next big step.
In addition to circumventing the need for laboratory synthesis of double-stranded RNA fragments, the RNA-silencing vector greatly extends the possibilities for practical application. The most important outcome of the new technique is that the vector induces persistent silencing of protein synthesis, whereas when synthetic RNAs are applied to the cell, the effect may be only transient. "There are experiments that just can't easily be done with synthetic double-stranded RNA," Shi said.
Guangchao Sui and Christina Soohoo, together with El Bachir Affar, Frederique Gay and Yujiang Shi all from the Shi lab, constructed a series of these vectors, each containing a short DNA template from a different normal gene. In each case, when the plasmid was transferred to cultured cells, the small interfering RNAs were made, and there was robust inhibition of specific target protein synthesis. Similar experiments were done in four different cell lines. In each experiment, target genes were silenced in 87 to 98 percent of the cells, regardless of the type of cell used. The results demonstrate that the technique is effective in a variety of cells and on a wide range of genes.
Harvard Medical School has more than 5,000 full time faculty working in eight academic departments based at the School's Boston quadrangle or in one of 47 academic departments at 17 affiliated teaching hospitals and research institutes. Those HMS affiliated institutions include:
Beth Israel Deaconess Medical Center
Boston VA Medical Center
Brigham and Women's Hospital
Cambridge Hospital
Center for Blood Research
Children's Hospital
Dana-Farber Cancer Institute
Harvard Pilgrim Health Care
Joslin Diabetes Center
Judge Baker Children's Center
Massachusetts Eye and Ear Infirmary
Massachusetts General Hospital
Massachusetts Mental Health Center
McLean Hospital
Mount Auburn Hospital
Schepens Eye Research Institute
Spaulding Rehabilitation Hospital
Contact: Judith Montminy, 617-432-0442, judith_montminy@hms.harvard.edu
Journal
Proceedings of the National Academy of Sciences