وضع عامل النمو المغلفه الخرز على أجنة الدجاج المرحلة المبكرة

Welcome to the Raburn Lab. I'm Matt Korn, department of Neurobiology and Behavior at the University of California Irvine. Today we're gonna take an egg, open it and place a bead that's been soaked in growth factor.

The wonderful preparation of a egg will allow us to put in a growth factor to change the fate of a number of different types of tissues in this early stage of embryonic development. Day two and a half, or stage 17. Okay, so we're here at, at the scope here.

I've got my eggs and all my tools. So go, go ahead and just pull an egg here and you can see where our tape is. Window seal.

I've got this nice little donut. It's just made out of artistic clay that you can get an art store. And I'm gonna take a really bad pair of five forceps.

These have been around for a long time. The tip tips are pretty dull, but they work great to just lift up the tape just enough so that window comes up. I can put those down.

I'm just gonna fold this over and tuck the egg, this window behind there like that. So now I can put it under the scope, make any type of manipulations. In this case, we're gonna be doing the beads, and I'm gonna go ahead and grab my India ink right now without the India ink.

You can see that there's a bit of a embryo disc right here, and it's off to the side a little bit. So if I just turn the egg, then I can see where some of the blood is forming a circle here. And I can go ahead and get my needle with the India ink.

And I'm gonna just come off to the side here, and I'm gonna go underneath the disc and there's an embryo. I don't wanna put it in too much enough to see the contrast of the embryo against the black. And then I'm just gonna go ahead and rotate this so that the roster part of the embryo is facing forward.

And what you can see here is the cephalon region. The neural tube comes back here and we have some somites that allows us to age it. And if I go ahead and zoom in, we can see that the heart has a very slow but consistent beating, which is great.

The heart was there, but not beating. We'd have to be a little bit concerned about whether or not this was a vital sample. So because there's no real thick membrane over it, I can just go ahead to my tungsten probe here.

I, and there's a thin membrane, and I'm just gonna move aside here so I can access the embryo. We place beads in, in the forebrain as well as the hind brain. And this one, I'm gonna be going for the forebrain.

Now, in order to get to it, I have to cut through a membrane. Now when they're younger, I can just use a tungsten probe and get in through the neural tube and I'll have access to the region where I want to put the bead. And now I'm gonna go ahead to my tungsten probe.

And I'm using two hands just to steady my hand. I'm gonna go ahead and maneuver the embryo. So once I get the bead in here, I can place it directly into the neural tube and up to to the forebrain.

Okay, so now that I have the the embryo open, I can go ahead and prep it and place the bead. So what I'll do is just stick my probe a little deeper and open up the neural tube, which you can see I've done there. I'll go ahead and zoom in a little bit closer.

I just change my focus a little bit here. And you can see there's a nice little opening there, but I haven't really put a hole above where the tail cephalon is, and that's because where the bead is gonna go is there. So I don't want a hole where the bead could float out.

I want it kind of a little bit back, so then I can put the bead in and push it forward. So now I'm gonna go ahead and get a bead. You can see that there's a lot of different sizes.

These are heparin acrylic, they're in PBS. I'm gonna go ahead and take a pair of 55 forceps. You can see that I'll go into the PBS and I'm just gonna find one what I like.

And once it's outta the PBS, it usually sticks to the forcep there, can see it there. Go ahead and cover the beads. Bring my egg back.

I'm gonna get the egg in focus. So I'm gonna go ahead and bring bead in on the 55 forceps. It's a little bit higher on the forcep that I like, but there it is.

And I'm just gonna kind of guide it over to where I made that hole in the neural tube. Once I get it into the neural tube, then it becomes easy to manipulate towards the front of the developing brain. So I'm gonna go ahead and take my tungsten probe, just push it.

When placing the bead in the cephalon, you have to be careful because what will happen is that as the embryo ages, it will begin to bend forward and then to the side. And a lot of the times what happens is when you're placing a bead in the forebrain telling Cephalon, it'll fall towards the beak because the beak forms on the ventral portion of the embryo where the brain is on the dorsal region. But this looks pretty good.

I'm fairly satisfied with this. I'm gonna go ahead, take a pipette with 10 microliters of sterile PBS, and I'm just gonna put a couple drops here. Okay, so now we can go ahead and close the egg.

And what I'm gonna do is just bring the window up here, up and over, and the egg isn't sealed yet. So what I want to do, so it won't dry out, is I'm gonna go ahead and take some of this plastic tape that we've been using and just cut a piece. It's about the size, just a little bit bigger than the window.

And I'm gonna stretch it just a little bit, and I'm gonna place it right over the window here and make sure it's nice and snug. So any of the holes that we've made, either when draining the albumin or cutting the window, they're sealed. So now the egg is sealed, it can't lose any of the umin, it won't dry out.

It should be able to age normally. So that's it for bead placement. Good luck with your chick experiments, and I hope you received the results you'd like.