Review of Research Paper: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells
The significance of this experiment is that it shows how siRNA suppresses the expression of genes in different mammalian cells. It was known previous to the experiment that dsRNA can trigger apoptosis in cells- this is an automatic defense mechanism that mammalian cells use to protect against the dsRNA possessed by viruses. The dsRNA can cause RNA interference when it is taken into the cell by a transgene or a virus. The dsRNA is then cleaved by ribonuclease III enzyme into 21-22 nucleotide siRNA's. The siRNA's joins a nuclease complex to form an RNA-induced silencing complex. This complex then cleaves and degrades mRNA. The question was, could transfecting the cell directly with siRNA produce RNA interference?
This experiment was performed to test whether siRNA's are capable of RNAi in mammalian cell cultures. (The idea to test gene silencing on mammalian cells sparked from a petunia-darkening experiment.) To do this they synthesized siRNA duplexes against genes that coded for sea pansies and two variants of firefly luciferases. Luciferase is used because it emits light so it's easy to see if the genes are turned on or off, and by what degree. The luciferase activities were recorded 20 hours after transfection and it was seen that the specific inhibition of luciferase was complete, which is similar to the results obtained for dsRNA. In mammalian cells where the reporter genes were more strongly expressed, the ability of the siRNA to completely suppress the gene was reduced.
So, what's with all these petunia flowers anyways?
Gene Suppression Within Plants
Plant scientists are always looking for approaches to over-express endogenous genes in plants in order to produce flowers with enhanced aesthetic characteristics or food products with greater nutritional value. A transgene is designed using molecular recombinant techniques and inserted into the plants genome. There are many different ways to insert the transgene including a bacterial host. The host can insert its own DNA into the plant. Harmful genes in the bacteria can be replaced with genes of interest, which produces a plant with an altered genome. Instead of overproducing the gene of interest, sometimes the transgene silences itself; this is termed "sense co-suppression".
Dr. Rich Jorgensen made an interesting discovery while trying to deepen the purple color of petunias when he introduced a pigment-producing gene under the control of a powerful promoter. The petunias were expected to turn deep purple, but they appeared variegated or white. "Co-suppression" had taken place because the introduced gene and the homologous endogenous genes were suppressed.
Materials and Methods
How do you silence a human gene?
This is how it was done:
Cells were cultured on a petri dish with a serum base. They were grown to form a monolayer...