February 10, 2004
Stanford Researchers Develop Fast Cheap Way To Silence Genes

A Stanford team has developed a way to do RNA interference (RNAi) on many genes in a way that is cheap and fast enough to allow much wider use in research laboratories.

STANFORD -- Sometimes the first step to learning a gene's role is to disable it and see what happens. Now researchers at the Stanford University School of Medicine have devised a new way of halting gene expression that is both fast and cheap enough to make the technique practical for widespread use. This work will accelerate efforts to find genes that are involved in cancer and the fate of stem cells, or to find genes that make good targets for therapeutic drugs.

The technique, published in the February issue of Nature Genetics and now available online, takes advantage of small molecules called short interfering RNA, or siRNA, which derail the process of translating genes into proteins. Until now, these molecular newcomers in genetics research have been difficult and expensive to produce. Additionally, they could impede the activity of known genes only, leaving a swath of genes in the genetic hinterlands unavailable for study.

"siRNA technology is incredibly useful but it has been limited by expense and labor. A better method for generating siRNA has been needed for the whole field to move forward," said study leader Helen Blau, PhD, the Donald E. and Delia B. Baxter Professor of Pharmacology. She said some companies are in the process of creating pools, or libraries, of siRNA molecules for all known genes in specific organisms but these libraries aren't yet available.

Pathology graduate students George Sen, Tom Wehrman and Jason Myers became interested in creating siRNA molecules as a way of screening for genes that alter the fate of stem cells -- cells that are capable of self-renewal and the primary interest of Blau's lab. The students hoped to block protein production for each gene to find out which ones play a critical role in normal stem cell function.

"I told them that creating individual siRNAs to each gene was too expensive," said Blau. Undaunted, the students came up with a protocol for making an siRNA library to obstruct expression of all genes in a given cell -- including genes that were previously uncharacterized. They could then pull individual molecules like books from a shelf to test each one for a biological effect.

The team had several hurdles to overcome in developing their protocol. The first was a size limit -- an siRNA molecule longer than 29 subunits causes wide-ranging problems in the cell. The key to overcoming this barrier was a newly available enzyme that snips potential siRNA molecules into 21-subunit lengths. A further step copied these short snippets into a form that could be inserted into a DNA circle called a plasmid. When the researchers put a single plasmid into a cell, it began churning out the gene-blocking siRNA molecule.

The group tested their approach by creating a handful of siRNA molecules to genetically disable three known genes. In each case, their technique generated siRNA that effectively blocked the gene in question.

Wehrman said this technique of creating siRNA molecule libraries could be widely used to find genes that, when disabled, cause cells to become cancerous or alter how the cells respond to different drugs. These genes could then become potential targets for drugs to treat disease.

A paper in the same issue of Nature Genetics described a similar way of creating siRNA libraries. "Having two unrelated groups working on the same problem shows there has been a real need for the technology," Blau said. The Stanford group has filed a patent for its technique.

Here is yet another reason why the rate of advance in biological research is accelerating. Better tools and techniques speed the rate at which experiments can be done and increase the amount of information that can be collected.

The abstract for the Stanford team's work is here. The other team the press release mentions is a Japanese team from the University of Tokyo. You can read their abstract here.

On a related note also read my recent post on the results of another team's effort to develop a technique to interfere with the activity of many genes at once using RNA interference: Massively Parallel Gene Activity Screening Technique Developed

Update Another report from MIT Whitehead scientist David Bartel and MIT assistant professor of biology Chris Burge on computational methods for finding a type of RNA called microRNA which regulates RNA expression.

CAMBRIDGE, Mass. (Jan. 28, 2004) – Research into the mechanics of microRNAs, tiny molecules that can selectively silence genes, has revealed a new mode of gene regulation that scientists believe has a broad impact on both plant and animal cells. Fascinated by the way microRNAs interfere with the chemical translation of DNA into protein – effectively silencing a targeted gene – scientists are exploring the role that these miniature marvels play in normal cell development and how they might be used to treat disease.

A critical component of understanding how microRNAs work in humans has been identifying which genes’ microRNAs silence and what processes they control. In a recent study, scientists identified more than 400 human genes likely targeted by microRNAs, taking an important step toward defining the relationship between microRNAs and the genes they target, including those linked to disease and other vital life functions.


In 2003, Bartel and Chris Burge, an assistant professor of biology at MIT, developed a computational method able to detect the microRNA genes in different animals. Using this method, they estimated that microRNAs constitute nearly 1 percent of genes in the human genome, making microRNA genes one of the more abundant types of regulatory molecules.

Bartel and Burge then set out to apply a similar approach to defining the relationship between microRNAs and the genes they target. Last month in the journal Cell, their labs reported that they have created a new computational method, called TargetScan, which does just that.

For each microRNA, TargetScan searches a database of messenger RNAs (mRNAs) – chemical messages that transcribe DNA into protein – for regions that pair to portions of the microRNA, and assigns a score to the overall degree of pairing that could occur between the microRNA and each mRNA. Those mRNAs that have high scores conserved in three or more organisms are predicted as targets of the microRNA.

Using this method, the team identified more than 400 genes in the human, mouse and rat genomes likely to be regulated by microRNAs. In addition, TargetScan predicted an additional 100 microRNA targets that are conserved in humans, mice, rats and the pufferfish.

According to Burge, 70 percent of targets predicted by TargetScan are likely to be authentic microRNA targets and the experimental data in the paper supports that a majority of their predictions are correct.

The take-home lesson here is that advances in the development of computer algorithms and the development of better tests and instrumentation are all accelerating the rate at which scientists can figure out systems of gene expression and genetic regulation in cells.

Share |      Randall Parker, 2004 February 10 11:02 AM  Biotech Advance Rates

back40 said at February 10, 2004 12:40 PM:

This is important. We have little understanding of regulation of gene expression though it is much more important than the genome itself. Some liken it the software than makes general purpose hardware so useful. These techniques may allow us to step the programs and come to understand their algorithms.

Wes Ulm said at February 16, 2004 12:05 AM:

Interesting stuff. I actually work in the RNAi field some myself, and it's such a fabulous technology. (Presenting some work for siRNA's used as anti-cancer agents at a conference in Singapore later this week.)

The amazing thing is that, if Andrew Fire and the other discoverers of RNAi hadn't been working in their own little tiny niche of C. elegans biology (where RNAi is especially active and there's no interferon response with larger double-stranded RNAs, which are chopped up by Dicer into the siRNAs to target the mRNAs to RISC, this whole phenomenon would've probably remained undiscovered for a decade or so. And then there were the plant biologists who were providing hints before that.

Luck and serendipity are a big part of science, but this is also why it's so important to keep funding basic science research that lacks any immediate apparent practical applications. Penicillin, bacterial restriction enyzmes, RNAi-- all similar stories, with a sharp-eyed postdoc or grad student noticing something the group wasn't seeking out initially.

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