"But RNAi didn't work in the vast majority of human or mouse cells because there are additional antiviral responses that recognize double-stranded RNA," said Elledge. "While the machinery to do RNAi is in mammalian cells, the antiviral machinery makes the introduced RNA toxic, and the cells die."
Researchers subsequently discovered that short segments of interfering RNA could be introduced into mammalian cells and remain unnoticed by the antiviral machinery, said Elledge. Furthermore, they discovered that the cell itself could be engineered to make interfering RNAs by introducing the gene for short hairpin RNA molecules that fold back on themselves to create a small RNA.
To construct a library of mammalian genes for short hairpin RNA molecules, Hannon and his colleagues first had to settle on an optimal design for a short-hairpin-RNA molecule. "We tested a lot of different things -- for example, the length of the hairpin, the loop structure, the structure of the transcript and what promoters to use," said Hannon. "And we arrived at an optimal structure for this phase of the science."
Hannon emphasized, however, "that set of parameters is something that is going to evolve continuously. There have been many advances over the last year in understanding of the biochemistry of RNAi. So, we are now constructing even more effective structures and even more effective delivery vehicles which will be built into future generations of this library."
Once an optimized basic design of the short hairpin RNA molecule was finished, the researchers then produced a library of genes for short hairpin RNAs that could target 9,610 human genes and 5,563 mouse genes. The genes chosen were those that were likely to b
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Contact: Jim Keeley
keeleyj@hhmi.org
301-215-8858
Howard Hughes Medical Institute
24-Mar-2004