Researchers at Howard Hughes Medical Institute (HHMI), Harvard Medical School, the University of Heidelberg and the Max Planck Institute for Molecular Genetics in Germany have demonstrated in the fruit fly Drosophila a general technique usable in any organism to simultaneously assay thousands of genes to determine whether each gene is involved in a particular aspect of cell function.
“A major challenge now that many genome sequences have been determined, is to extract meaningful functional information from those projects,” said HHMI researcher Norbert Perrimon, who directed the study. “While there are a number of analytical approaches that can measure the level of gene expression or the interaction between proteins, ours is really the first high-throughput, full-genome screening method that allows a systematic interrogation of the function of every gene.”
The technique uses double-stranded RNA made to match every known gene in the target organism that is of interest. The double-stranded RNA causes a phenomenon called RNA interference (RNAi) which blocks the action of the corresponding RNA strand which gets made from each gene. Nornally cellular machinery reads a gene in the DNA and creates what is called messenger RNA (mRNA) which has a matching sequence to that gene. Then that mRNA is read to make proteins. But dsRNA prevents that step and therefore blocks the creation of proteins. This effectively blocks the gene from having any effect and then the automated assay system of these researchers watches what the effect is on cell growth or on whatever other aspect of cellular activity the system could be set up to measure.
The screening technique developed by Perrimon and his colleagues builds on methods developed in one of the hottest areas of biology, RNA interference (RNAi) research. In RNAi, double-stranded RNA (dsRNA) that matches the messenger RNA produced by a given gene degrades that messenger RNA — in effect wiping out the function of that gene in a cell. RNAi is widely used as a research tool to selectively erase the cellular contributions of individual genes to study their function.
In their mass screening technique, Perrimon and his colleagues first created a library of 21,000 dsRNA that corresponded to each of the more than 16,000 genes in the Drosophila genome. They then applied each of these dsRNA molecules to cultures of Drosophila cells and assayed how knocking down the function of a targeted gene affected cell numbers in the cultures. This basic measure, said Perrimon, revealed genes that are not only involved in general cell growth, but also in the cell cycle, cell survival and other such functions.
The researchers then selected 438 genes for further characterization. The degradation of these genes produced profound affects on cell number. “Out of this subset, we found many that produced proteins involved in general metabolic processes such as the ribosomes that are components of the protein synthesis machinery,” said Perrimon. “But we also found genes that are more specific to cell survival.”
According to Perrimon, only 20 percent of the genes that were identified had corresponding mutations — an important characteristic for studying gene function. “The classic approach to studying gene function is to identify mutations in genes and select those that produce interesting phenotypes that yield insight into function,” said Perrimon. “But this approach has never really given us access to the full repertoire of genes. With this high-throughput technology, however, we can study the function of a complete set of genes. We can systematically identify all the genes involving one process.”
The technique can be used to screen for genes involved in intercellular communication, cancer cell proliferation, and other cellular activity. Combined with drug screening the technique can accelerate the search for drugs that operate on particular cellular pathways and processes.
The RNAi assay will contribute to the screening of new drugs, he said. “One exciting aspect of this approach is that we can combine our assay with screening of potential therapeutic compounds,” he said. “One of the big problems in the pharmaceutical industry is that researchers may discover pharmacologically active compounds but have no idea what their targets are in the cell. However, it would be possible to perform coordinated screens — one for compounds that interfere with a target pathway and an RNA interference screen for genes that act in that pathway. This correlation would allow you to match the compounds with the proteins they affect in a much more useful way.”
One can see by reading between the lines here how this technique has to be built on top of a lot of other existing tools that automate the creation of needed components. There has to be a fairly automated existing technique to generate all the different kinds of dsRNA strands used in this techinque. Also, the technique must rely on an automated tool for, feeding cells, measuring cell growth, and doing other steps in this process.
Results of studies for new ways to treat diseases or discoveries of ways that genes and cells work get a lot of press attention. But the ability to automate and therefore accelerate massive parallel screening and manipulation of genes, proteins, and other parts of cells is what makes possible the faster rate of discoveries of disease causes and disease treatments. Cells are so complex with so many pieces, subsystems, and types of interactions that only with the development of massively parallel techniques can we hope to fully figure out how cells work and how to cure most diseases in the next few decades.
|Share |||Randall Parker, 2004 February 05 12:13 PM Biotech Advance Rates|