发育研究的窗口鸡蛋

Welcome to the Kramer Lab. I'm Matt Korn, department of Neurobiology and Behavior at the University of California Irvine. Today I'll be showing you how to prep chicken eggs for a variety of manipulations, including bead placement, the injection of dyes, or in ovo exploration.

So without any further ado, let's get started. Okay, so we're gonna go ahead and clean the egg. Reason being is there's a lot of things that could be put on in the incubator.

We're gonna be sealing this with tape. We don't want any small bits getting around where the window's gonna be getting the egg and and increasing the chance that the embryo will die by the time we want to take it out. So if you saw from the incubator, the the eggs are sitting with the big side facing up.

So what we're gonna do is we're gonna go ahead and take the egg and just turn it to the side. And that is gonna do two things. One, our window's gonna be right here, and secondly, the embryo needs to float to the top so we can make any type of manipulations.

So I'm gonna set out a few here from the rack that was in the incubator to a cardboard egg holder. This is actually the piece that comes in the box when the eggs are delivered. So for today, we're gonna go ahead and use it just to hold the eggs.

Now the arrangement is, is up to you. I like to do it this way because I can access all the eggs at any time and none of em are touching each other. In case I were to find one that was loose that had a crack and were to drop it, they all kind of have a little bit of space from one another.

So I'm gonna go ahead and take my gauze here and just take out a nice stack like this and split it in two. I'm gonna take 70%alcohol here and I'm just gonna fill it up until I can kind of feel the bottom getting damp. And that's cause I'm gonna use this whole stack to swab all the eggs.

So then I'll just take a couple off the top, go ahead and just really do the top but in effect almost get around the whole egg here. And after this is done, the alcohol dries pretty quickly and then we can go ahead and put tape on it. The tape will stick nicely and by the time that I finish a set, then usually the first one is dry and we can proceed onto the next step in our egg preparation.

So again, place that there back in the trash and we're ready to go to the next step. Okay, so now that all our eggs have been swabbed with the alcohol, we can go ahead and make a drain hole on the big side of the egg, the the base of the the egg. And this will allow us to go in and extract some albumin because if we don't extract the albumin then the embryo doesn't sit properly.

We can't make any manipulations. So this just involves me taking a blade and take this plastic tape. It's a little bit of elastic, which is great.

Not only does it allow us to expand over a hole if it's a little larger than we'd like, but when we actually cover the entire egg, we're gonna need to stretch this. So the area where the window's gonna be is entirely covered. So I'm just gonna go ahead and cut the piece off like this and then make about a millimeter strip.

And then from this I can make about a millimeter square piece of tape. And then from this I'm just gonna go ahead and place it just above to the left of the date. Just put it right there and you can see it.

It's sticking very nicely. Then I can come back to the strip that I already made, cut another millimeter piece of tape off. And again, just place it to the left of the number there, which is actually the top of the egg.

Now that we've turned it sideways, it's to the right. And one more piece here. Now I'm not wearing gloves because wearing gloves will make this tape stick to the gloves and I've washed my hands.

I'm not really concerned about anything else contaminating the active at this point. So I'm gonna go ahead and take a pair of scissors with the sharp point. I'm gonna go in the center of where I placed the tape and I'm just gonna turn it in a counterclockwise clockwise motion until I fill it break just a little bit.

And you'll see that there's a small hole there. Now the hole doesn't need to be very big because once we get this syringe in there, it'll make the hole big enough. But this is so we're not going straight in with a needle and trying to break the egg with that because oftentimes that'll lead to the whole side of the egg breaking apart.

Then we can't use the egg anymore. Okay, so now I'm gonna go ahead and drain the egg. We've got that nice little hole on the side and I'm gonna use this 10 milliliter syringe and a fairly large gauge needle to go ahead and extract the albumin.

Now I want about three or four milliliters, definitely more than three, not much more than four and a half or five. This will allow our embryo that's now sitting on the top to fall a little away from the shell. And then we'll be able to make our manipulations.

So by going in at a 45 degree angle, I'm underneath the yolk. And then I just wanna turn it a little bit to my right just to sneak under there and you can kind of feel the needle tip just touching the bottom of the egg. And I'm gonna go ahead and extract just about three or four and it comes out pretty nicely.

I'm gonna go ahead and put that albumin into my waist bucket here and now I can continue with the other eggs. Okay, so I'm gonna go ahead and continue. And you'll see here that the hole that I've made for the draining is a little bit larger than I'd like, but that that turns out to be okay.

What I wanna be concerned about is that as I'm sticking the needle in that I don't crack it anymore. So I'm gonna go ahead and do that. Okay, so I can already see that the egg has a bit of a crack in it.

Now small cracks are okay, but you need to be concerned when it extends to another plane of the egg. And in this case it does. You can see that from the hole.

It extends pretty much to the top. And in fact, this is where our window's gonna sit. So unfortunately for this egg, we're gonna go have to and discard this one.

And there's nothing special about discarding it. You don't need to write up a particular protocol or anything. Just gonna go ahead and put it in the waste bucket.

We use these black trash cans to designate them as animal waste, but other than that, it's already been taken care of. We can move on to the next egg. Okay, so now that we've drained all our eggs, we can go ahead and cover the drain hole.

Now this is important because if we don't the inside the incubator, we can get a tremendous evaporation of whatever is left of the albumin. And then an egg that is dry is an egg that is dead. So we're gonna go ahead and just take about the same size of tape and place it over the hole and we won't have to worry about going into this hole again.

In some cases, if you decide you would like to try draining more albumin from an egg, once it's opened, this hole acts as a bit of a sealed area that you can then stick a needle in and you won't lose a whole lot of albumin. Okay, does it? Okay, so now we're gonna go ahead and cover the eggs with a nice size piece of tape.

And I kinda liken this to putting a piece of tape over a balloon before you put a little pin in it. Cool thing is the balloon doesn't pop and in this case a little tape over the egg. The egg doesn't crack in multiple places and then the window can then be sealed and then when the window shuts, because we've used tape it, it kind of creates a nice little seal and we don't need to worry about any evaporation.

So you can see the size of tape is, it's probably the biggest that I've cut so far, but even then I'm gonna go ahead and stretch it just a little bit and really just tug on it, a little bit of tension there and place it over the egg. And I'm gonna come around the back here and stretch it just very carefully over the egg so that there's no creases right over where my window's gonna be. And you can reset the tape a little bit.

It's, it's not sticking so much that you have to worry about. Once it's stuck, it's stuck. And you can see this general area here is where I'm gonna go ahead and cut the window.

Okay, so now that we have tape on all the eggs, we can go ahead and cut a window on the top. This can be the most difficult part and often this is where you'll lose the majority of your eggs. So I'm just gonna go ahead and give a good wipe to my scissors with gauze that had alcohol on it and then go ahead and just dry it.

Alcohol pretty much dries off by itself, but you don't want alcohol going straight into the egg. So I'm gonna just go in the middle of where the tape is and again, use that clockwise counterclockwise motion until I hear it crack a little bit. And then I know that my blade is in.

And then I kind of wanna move my scissors out so they're almost flat against the surface of the egg. And then just make tiny snips until I can get the entire piece of scissor in there. And I'm gonna come back the other way once I've made it about a half circle and I don't want to complete the circle cause I want a window, but then I can just go ahead and open the egg.

And right now it looks like there are about the age that I expected them to be, don't have a whole lot of blood vessels, but there is a bit of blood around where the embryo is sitting primarily from the delion region that the blood has now started and the heartbeat has gone. So I'm just gonna shut that, just let it sit on top like that and come around again and I'll go ahead and shut the windows. And again, just, just flat to the surface and back the other way.

And okay, so we've got a couple bubbles here and it turns out that the embryo is off to the side. Now there's two things we can do right now. If we have plenty of eggs and we're not concerned about using every single one, we can go ahead and toss this.

Or what we can do is we can shut the window and just set it so that the embryo will kind of float up to the top where we want it again. And by the time we get to it in our manipulations, it might be where we want it. If not, we can discard it at that time.

So what I've done right here is just set it at an angle. You'll have a little bit of albumin runoff, but it'll help that embryo get back to where it needs to be. Okay, so now that we have all these windows, we, we need to make sure that they're closed and we just want em closed before we make any manipulations.

And then we can go ahead and reseal the egg. But if we have an open window, sometimes the yoke can slip out. If you know the egg just tips a little bit when you're walking from one room to the other, you don't want the whole thing falling out.

So all I'm doing is cutting another millimeter long, or I'm sorry, millimeter wide, probably about three, two or three millimeter and length piece of, of the plastic tape. And I kind of like to put it there just on the middle. And if I can pull it down in front of the egg, then it kind of makes it tight.

And you can see where the window fits, right where the other tape was, where we cut the window and it makes it pretty sealed for this time. I mean, we're gonna go ahead and make some manipulations in in just a minute, so it doesn't need to be entirely airtight. So that's how you prep and window your eggs.

So good luck with your experiments.