My Master’s Project

All labwork is overseen by the disembodied head of Muppet lab assistant, Beaker.
All labwork is overseen by the disembodied head of Muppet lab assistant, Beaker.

I have begun my Master’s project in earnest and the goal is slightly different than what I’d been doing before.

First, I’ll repeat myself. I’m a biologist and I work with introns in C. elegans. C. elegans is a type of nematode worm that naturally lives in soil or on rotting vegetables. It is also one of the most widely used model organisms for biological research.

Worms! Ew! Gross!
Worms! Ew! Gross!

Introns are unused sections of genes. You’re probably aware that DNA is in our cells and contains the instructions for how an organism functions. The human genome contains around 25,000 genes and those genes are split into two parts, introns and exons.

Exons are the part of that gene that are actually used to produce things in your cells, while introns are spliced out and removed. So why are introns on there at all if they’re removed?

Well it turns out that some introns increase expression of the genes they’re in. My project looks at how placement of those enhancing introns affects expression.

Experiments in plants have shown that an enhancing intron works best when it is placed near the start of a gene. Experiments in C. elegans have suggested that, but no experiment has outright proved it. My project will hopefully do that.

I’m measuring the expression of genes according to how introns affect them, so I get to pick which gene to use. When picking a gene like this scientists often pick what are called reporter genes. The expression of these types of genes is easy to measure, often because they have produce light or fluorescence of some kind. The light tells you whether the gene is on, but also at what level it is turned on based on how bright the cell is.

Previously I was using a reporter gene called GUS. GUS is an enzyme that digests a specially prepared sugar, releasing a blue chemical that was attached to that sugar. The blue chemical is then visible to the naked eye.

There were a number of problems with that experiment though. First, adding the sugar chemical to the worms was a pain, taking about three days to set up and look at. Plus, the blue color was difficult to measure precisely because most of the machines in the lab are set up to measure red or green colors, not blue. Finally, GUS is traditionally a reporter gene for plants, not C. elegans. This could’ve been introducing other problems that we couldn’t easily identify. Thus the use of the GUS reporter gene has been scrapped in favor of another reporter gene.

I’ll be using Green Fluorescent Protein (GFP) as my reporter gene now. GFP is widely used in C. elegans and many other organisms. The protein created by the GFP gene glows green when you shine a red light on it. Very easy to see and measure. None of that three day procedure for GUS. I just pop the worms under the light and take a look.

Why weren’t we using this procedure before if it’s so easy? Two reasons!

Reason number one: C. elegans won’t express GFP without introns in the gene. So does that mean we proceed and hope one intron is enough or do we add the standard amount of introns to get expression? I’ve decided to see what the GFP looks like with the standard introns scientists put in it for C. elegans and without them. I’ll also be testing with an added intron. The whole thing is a little complicated so here’s a diagram to explain.

Here are the constructs I've been creating. The wide parts are exons and the thing parts are introns. The green bands are the GFP which will glow green in the worms. The white bands are a scaffold which allow the worms to express GFP
Here are the constructs I’ve been creating. The wide parts are exons and the thing parts are introns. The green bands are the GFP which will glow green in the worms. The white bands are a scaffold which allow the worms to express GFP.

There are eight different constructs I’m making. They are a combination of three different features that are present or not. Are there introns in the first GFP? Yes or no? The second GFP? And is the Unc54 intron there? This allows us to control for the positional effect the standard introns in C. elegans GFP.

Reason number two: Those eight constructs above? Those aren’t made yet! All the GUS constructs were made when I started the project. I’ve been working on making the new constructs for a few months. It could take a few more months to finish.

So my project is to make those constructs, put them into worms, and then see what the worms look like. As I perform these steps I’ll make more posts about what work I’m doing in lab and why its so cool.

-Mister Ed

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Injecting Worms

This is what my computer captures under my microscope when I inject a worm.
This is what my computer captures under my microscope when I inject a worm.

I gave an extra post about one of my jobs. It seems fair to cover the other job as well at some point!

I study introns in C. elegans worms, but how do I get the specific introns in the worms?

I need introns in specific placements in specific genes in order to study them with scientific accuracy.

The gene we are studying is simple. If the worms are put in a solution called X-gluc, they turn blue.

Based on where our enhancing intron is in the worm we expect it to turn more blue if the intron is closer to the start of the gene or less blue if it is near the end.

So I have these genes that I’m putting into the worm. They get in by injecting them like you see in the picture.

The needle of DNA is aimed at the gonad of the worm.

C. elegans worms are hermaphrodites. They contain sperm and eggs and they self-fertilize.

The worms are “male” at first, producing a bunch of sperm.

Later on they produce eggs and then they fertilize their own eggs with the sperm stored in their body.

Since they contain both genitalia the whole area is referred to as the gonad.

I aim my injection at the gonad, hoping that the DNA I’m injecting will get into the fertilized eggs.

Then the injected worm is put on a plate with lots of food and I hope that its babies will have the injected DNA.

But I don’t test for “blueness” immediately.

When a worm is first injected, the DNA is inside its cells, but not necessarily integrated into the cell’s chromosomes. I need the DNA to be a part of the chromosomes.

There are only two genes in the mix of injected DNA that will integrate. One gene is the blue gene, called GUS. The other gene is called unc119.

Unc119 is to “recover” the worms.

The worms I inject lack unc119, which is a normal gene for worms.

In a natural wild-type worm unc119 aids the development of the worm’s neural network. Without it, the worm has poor neural connections and has a lot of trouble even moving around and eating.

So the first way I test a successful injection is by looking to see if the babies of the injected worm are moving around normally or flopping around.

The normally moving ones were successful and now have unc119. They are “recovered” back to their natural wild-type state.

The floppy crippled ones did not have a successful injection. Either I missed the gonad, I didn’t inject enough DNA, or the eggs that got my injection didn’t fully germinate.

There are other markers I use to see if an injection was successful, but I’ll get to those later!

-Mister Ed

Worms and Introns

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I have two jobs right now. One of them is an internship working on introns.

Introns are part of your genes, but they’re a strange part.

Imagine your genes are like a TV show. There are parts you watch and there are the commercials that you mute or ignore.

When the TV show comes out on DVD or Netflix the commercials are removed.

Genes are split up into watchable parts and commercials too. The watchable parts are called exons and the commercials are called introns.

When DNA makes RNA the introns are removed from the code, just like when a TV show is released on DVD the commercials are removed.

For a while scientists thought that introns did nothing for the genetic code of an organism. Introns were just useless DNA trash.

That changed in the late 1980s when some introns were found to enhance the expression of genes.

Some genes have what are called enhancing introns that increase the expression of that gene. This is called intron mediated enhancement (IME).

If you take an enhancing intron from one gene and put it into another, then the other gene will create more RNA and thus more proteins as well.

So enhancing introns increase expression of a gene, but not much is known about why. The lab I work in is one of the few that studies this process to try and figure out the specifics.

Most intron research right now is done in plants. I’m trying to extend that research to animals by using worms.

The worms I use are called C. elegans. They’re only 1mm long and are commonly used for research projects around the globe.

My lab previously discovered that enhancing introns in plants work best near the beginning of a gene.

My project is to see if the same holds true for C. elegans.

I’ll also be looking at whether an intron that is enhancing in plants is also enhancing when out into a gene in C. elegans.

That’s all for now!

-Mister Ed