Gene knockdown by RNA interference
The complete genome of Tribolium has been sequenced. This means that we know exactly from which bases the DNA is build and in which order. Although it is good to have this information, it does not tell us anything about the function of certain parts of the DNA. I wrote earlier that protein is made from RNA and that RNA is made from DNA (see DNA, RNA and protein). Many different proteins can be made from DNA. A piece of DNA that codes for a specific protein is called a gene. We humans have for example approximately 25000 genes. But what is the function of all these genes? To answer this question there are several techniques available to us scientists. One of these is “RNA interference”. This technique induces the destruction of RNA which prevents the synthesis of protein. In the absence of this protein we can look for clues on the function of this protein. If we for example knock down the gene Ultrabithorax in the fruit fly, we get a fly with 4 wings instead of 2! (see picture) If we on the other hand knock down the gene Antennapedia in the fruit fly, we get a fly with legs where its antenna should be! (see picture) In this way we can see that these genes are involved in the development of the wings and antenna. Knocking down genes is possible with a couple of different techniques of which RNA interference is one. With RNA interference we make use of the immune system of the insect (here Tribolium). In short, we inject the beetle with double stranded RNA, the immune system recognizes this and will cut all the RNA with the same sequence into small pieces. The immune system responds in this way to double stranded RNA because many viruses carry double stranded RNA.
Many viruses use double stranded RNA as their heritable material instead of DNA. The rotavirus, which causes vomiting and diarrhea in children, is an example of a double stranded RNA virus. Once a virus has successfully entered the body, it will try to infect cells. A double stranded RNA virus consists of a mantel of protein with double stranded RNA inside. When a virus is able to infect a cell it will copy its RNA and produce new proteins for its mantel. The mantel will be assembled inside the host cell and loaded with a single strand of RNA. The second strand of RNA is made inside the mantel. This new virus is ready and will leave the cell again (which often causes the death of the cell). Luckily, the immune system is able to detect double stranded RNA. When the immune system recognizes double stranded RNA it will cut the RNA into small pieces. But it will not only cut the piece of viral double stranded RNA into pieces, it will cut any piece of RNA which has the same sequence of bases. This mechanism prevents the duplication of the virus inside the cell (see the figure below). This mechanism against virus infections is what we use for RNA interference.
When the immune system recognizes a virus by its double stranded RNA, all RNA molecules with the same sequence are cut into small pieces. Because the RNA is cut into pieces, protein can no longer be made from it. So if we inject double stranded RNA with the same sequence as the gene we want to knock down, the immune system will cut all RNA molecules with that sequence into pieces, even the ones that are made by the organism itself (see the figure below).
In Tribolium, this RNA interference works throughout the body. So no matter where we inject the double stranded RNA, the knock down will be in the entire body. If we inject double stranded RNA into female beetles we will knock down the gene in that female beetle, but we will even knock this gene down in the eggs that she will lay! This is called parental RNA interference and gives us the ability to assess the function of genes during embryonic development.
So with this technique we can knock down genes and assess the function of genes (of the proteins the normally make). Take a look at my “techniques” page to see how RNA interference in Tribolium is done practically.
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