I now have my very own box in the freezer in my project lab. (It has a bright green name sticker on it, because I feel I should keep up my reputation for liking shiny things :P) I feel like I belong there, finally!
Okay, that's an overstatement, but it does feel like my project is finally starting to go somewhere. So far I, and the other girl doing her third year project in the same lab, have spent five weeks doing Western blots over and over again, with various different protocols and equipment and extracts, and to a certain extent it felt like going around in circles. Now, however, we finally have a set up that works well, and tomorrow, perhaps, touch wood, we will have some results.
What I'm actually doing is looking at the effects of reducing the levels of certain proteins in C. elegans, a microscopic nematode worm (they're actually kinda cute, see above. At least I think so...). These worms are useful because there's a very simple, reasonably fast, and cheap method to reduce the levels of a specific protein in them by using RNA interference (RNAi). The worms eat bacteria, and it's possible to make bacteria that carry a gene for a "small interfering RNA". These do what they say on the tin, basically! They produce small RNA molecules (which aren't translated into protein, unlike the RNA from most genes), which interfere with the translation of a specific protein. It's possible to make RNAi molecules to target any gene and reduce the amount of the corresponding protein.
Then, you feed the bacteria carrying these RNAi molecules to your worms, and wait. It takes a few days to have an effect. I will probably have to repeat this step in my project, because it doesn't look like the effect is strong enough yet :(
The proteins I'm looking at are all histone methyltransferases (aka HMTs), enzymes that add methyl groups to the histone proteins that help to package DNA. Changing the methylation of histones changes how accessible the DNA is, and can alter gene expression. It's possible to see whether the amount of methylation on the histones changes directly, by using an antibody that detects methylation in specific places. I'm looking at a mark on histone H3 which is associated with transcriptional repression. I have some worms which lack an HMT known to add this particular mark, and I'm looking to see if using RNAi on various other genes that might add this mark has any additional effect.
So far, the answer is... maybe. *sigh* As always, results need replicating! So far I've only been able to look at 6/8 genes; I'll get results from the other two tomorrow and then see which look most promising, or whether I should just try again and see if I can get a stronger effect from the RNAi.
Okay, that's an overstatement, but it does feel like my project is finally starting to go somewhere. So far I, and the other girl doing her third year project in the same lab, have spent five weeks doing Western blots over and over again, with various different protocols and equipment and extracts, and to a certain extent it felt like going around in circles. Now, however, we finally have a set up that works well, and tomorrow, perhaps, touch wood, we will have some results.
C. elegans - an adult and a couple that have just hatched. Source.
What I'm actually doing is looking at the effects of reducing the levels of certain proteins in C. elegans, a microscopic nematode worm (they're actually kinda cute, see above. At least I think so...). These worms are useful because there's a very simple, reasonably fast, and cheap method to reduce the levels of a specific protein in them by using RNA interference (RNAi). The worms eat bacteria, and it's possible to make bacteria that carry a gene for a "small interfering RNA". These do what they say on the tin, basically! They produce small RNA molecules (which aren't translated into protein, unlike the RNA from most genes), which interfere with the translation of a specific protein. It's possible to make RNAi molecules to target any gene and reduce the amount of the corresponding protein.
Then, you feed the bacteria carrying these RNAi molecules to your worms, and wait. It takes a few days to have an effect. I will probably have to repeat this step in my project, because it doesn't look like the effect is strong enough yet :(
The proteins I'm looking at are all histone methyltransferases (aka HMTs), enzymes that add methyl groups to the histone proteins that help to package DNA. Changing the methylation of histones changes how accessible the DNA is, and can alter gene expression. It's possible to see whether the amount of methylation on the histones changes directly, by using an antibody that detects methylation in specific places. I'm looking at a mark on histone H3 which is associated with transcriptional repression. I have some worms which lack an HMT known to add this particular mark, and I'm looking to see if using RNAi on various other genes that might add this mark has any additional effect.
Repression/induction and changing histone modifications. Source.
So far, the answer is... maybe. *sigh* As always, results need replicating! So far I've only been able to look at 6/8 genes; I'll get results from the other two tomorrow and then see which look most promising, or whether I should just try again and see if I can get a stronger effect from the RNAi.
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