HH 스테이지 10 치킨 태아의 비켜 Electroporations에서

The chick is a good model system because the embryos are very amenable to manipulation and their large size also makes them much easier to work with than other embryos. Chick electroporation experiments also allow for the screening of a large number of genes in a short period of time. Hi, I'm Marissa Blank.

I'm a graduate student in the lab of Kathy Millon in the Department of Human Genetics at the University of Chicago. Today I'm going to be talking to you about how to perform chick and ovo electroporation, and we will be electro hamburger and Hamilton stage 10 chicken embryos, which is about two days of embryonic development in chick. The procedure is performed in our lab routinely, and usually what we are studying is the effect of either overexpression or knocked down of genes in the spinal cord because we're interested in genes that are involved in development of the central nervous system.

So the technique that I will be showing to you today in ovo electroporation involves several steps. The first step will be windowing the egg. The second step will actually be injecting DNA into the lumen of the neural tube.

And the third step will be using an electric field to push the DNA into one side of the neural tube. And then I will show you how to close up the eggs and we'll put them back in the incubator. Okay, before we get started, I'd like to tell you a little bit about proper egg handling.

So the eggs will be purchased from a chicken breeder and they'll come fertilized. Because these eggs contain embryos, you wanna be careful about the way you handle them. So prior to incubation, the eggs should be kept at 13 degrees.

For this purpose, we use a small wine cooler, and then once you're ready to incubate the eggs, they should be allowed to reach room temperature before they're actually put into the incubator. And then once they're put into the incubator, the incubator should be maintained at a hundred degrees Fahrenheit, which is about 57 degrees Celsius. It's also important that the incubator is humidified Humidity is really important for proper embryo development.

The first thing that you do to prepare these eggs for electroporation is to window them. And the first step in this process is to remove a little bit of the albumin from the egg. So I put a small piece of tape on the end of the egg, and I'm going to take a large bore needle and puncture a hole in the end, and then going to be very careful and point the needle down toward the bottom of the egg so as not to disrupt the embryo or the yolk.

And I'm going to use the syringe to remove about five milliliters of albumin. I will then reseal the egg just using another piece of scotch tape. And for electroporation, scotch tape is a good tape to use because it is non-toxic.

The next step is to place another piece of tape over the top of the egg where the window is being made. It's important to keep the eggs on their side while you're incubating them because the embryo will float to the top of the yolk. And so in this egg, which has been kept on its side, the embryo is actually right around here.

I've marked the top of the egg prior to putting them into the incubator so that I know where the embryo was going to be before I cut open the egg. So now using a pair of curved scissors, I'm going to make a small hole and then cut a window. You wanna cut a window that's as small as possible to allow you to work, and you can always make it larger later if necessary.

So now that we've windowed the egg, we are ready to proceed to the actual injection and electroporation. And for that, we're going to move to the microscope. So in order to visualize the embryo, I will be injecting some India ink underneath the embryo.

This is actually something that we don't do on a regular basis in our lab. Once you've done these experiments, you'll actually be able to see the embryo probably without contrast, but it is a little bit hard to see. So as the ink spreads out underneath the embryo, you should start to be able to see the neural tube quite clearly.

So the neural tube is this lighter colored region in the middle of the embryo and the way the embryo is oriented, currently, the head is facing away from me up at the top. This stage is also known as the hammer head stage. You can probably tell by, because the embryo does look like it has a hammer head.

So when injecting, I usually turn the embryo so that the head is facing toward me. And when I inject the embryo, what I will be aiming for is the region that is right below where the head sort of expands. So I'll be focusing, I'll be pointing my needle right about here and injecting at this stage.

You can also see somites forming along the sides of the neural tube here. Okay, so again, the head, and this is the neural tube, which will become the spinal cord. So now we have our embryo ready.

We're going to actually do the injections. And so the tools that you'll need to do the injections are first glass capillaries. We have these glass capillaries and a capillary pull pulls them into a glass needle.

We then take this glass needle. And in order to get the proper aperture for injection, we need to break the tip off the needle. This can actually be done either under the microscope or just by eye.

And so I usually just use a standard pair of number five forceps, and then I break the tip off the capillary. It's possible to break the tip off too far back or too high up, and to make the aperture not the right size. If you make the aperture too big, you'll be able to tell, because when you try to draw liquid into the capillary, there will be no resistance.

So you can usually tell if the aperture is the right size by the amount of resistance you get when pulling cap liquid into the capillary. The other piece of equipment that we use is a mouth pipette. So the mouth pipette is composed of three parts.

There is a mouthpiece, a piece of either latex or tai on tubing, something flexible, and then the part that actually holds the capillary. And so you'll just take the needle, insert it into the rubber gasket to hold it. So it, you also need to be kind of careful with these glass needles because they are very sharp, and if you stab yourself, you'll break off a piece of glass and leave it under your skin and that kind of hurts.

So I find that the best tube to hold your plasma DNA with fast green dye is a small PCR tube. So I'm going to suck up some dye into the capillary right now.Okay. And when doing this, be careful not to touch the bottom of the needle to the bottom of the tube because you will break it and you'll have to start over again.

Okay, so now the needle is loaded. We're ready to inject. So I have the embryo now oriented under the scope so that the head is facing toward me.

And I'm going to take the glass needle containing the fast green dye as well as the DNA and using a shallow angle. Put the tip into the neural tube into the lumen of the neural tube. And you need to be careful not to actually go through the other side, because then the dye will be injected under the embryo.

So I now have the embryo oriented under the microscope so that the head is facing toward me. I'm going to take my needle filled with fast green dye and DNA and using a shallow angle. I'm going to puncture the neural tube so that I can fill the lumen of the neural tube.

And when you're doing this, you need to be careful not to actually go too far and push through the other side of the neural tube, because then you'll be injecting your DNA underneath the embryo. You can see now that the neural tube is filled with this fast green dye a little bit if it has actually leaked out under the embryo. Ideally, you don't want that to happen.

You just wanna have the neural tube filled, but you should be able to fill the neural tube from the head all the way down to the tail. So now that we've actually injected the DNA into the neural tube, we are ready to do the electroporation itself. And the electroporation apparatus is what's right next to me here.

So it consists of several parts. The first part is the platinum electrodes. They're held in an electrode holder, and that's held by a micro manipulator that can be used to raise and lower the electrodes while maintaining their proper position.

The electrodes are attached to cables, and the cables are attached to a pulse generator. And the pulse generator is used to generate short pulses of electricity between the electrodes to actually force the DNA into one side of the neural tube. When we pulse the embryo, we generally use a 25 volt pulse.

We do five pulses that last 50 milliseconds, and that's over a period of five seconds. So it's just five short pulses of electricity. So now we have the electrodes positioned in the microm manipulator right above the embryo.

I'm going to gently lower the electrodes onto the embryo so that they are running parallel to the neural tube. Then I'm going to apply a little bit of media to help with the conductance of the electricity. And then actually pulse the embryo Rio.

You can see that there are bubbles forming on the electrode. That's a good sign that indicates that the electrode person actually worked and you made the connection that you needed to make. And now I'm just going to gently, gently pull the electrodes up from the embryo, and now the embryo is ready to be sealed up.

Now I've shown you how to electro parade the egg. The last step is also quite important, and that's sealing the egg back up so that it can continue to develop. So as I mentioned before, humidity is very important for the further development of the embryo.

And so you need to be sure that you've sealed the embryo properly. So to do this, you just take another piece of tape, put it over the hole, but be very careful to make sure that you have completely sealed the piece of tape around the edges so that you don't lose any humidity within the egg. And then the egg just needs to be stuck back into the incubator and allowed to develop until the stage of which you want to analyze it.

So in conclusion, today, I've shown you how to ate hamburger and Hamilton stage 10 chicken embryos, and specifically how to ate the neural tube of these embryos. The critical aspects of the protocol that I've just shown you are first windowing the embryo without damaging it. Second, injecting DNA into the neural tube again without damaging the embryo.

And third, the proper placement of the electrodes, which is critical to actually getting the DNA where you want it during the electroporation step. Finally, I'd just like to remind you that it is important to properly seal the egg before you put it back into the incubator. When we ate DNA, we use a plasmid that has a GFP reporter, and we generally use that GF reporter as an indicator of expression of our gene of interest.

Following electroporation, you can usually expect to see GFP expression within six to 12 hours. However, this doesn't necessarily mean that your protein of interest has been expressed to the optimal levels. So when you're doing these experiments, you may want to analyze the embryos between one and two days following electroporation in order to get the optimal expression level.

Good luck with your experiments, and thank you for watching.