E15マウス胚の子宮内での脳室内注入とエレクトロポレーションで

Hi, I'm William Tis from the Krete Lab. Today, myself and others are gonna be showing you intrauterine injections of E 15 mouse embryos. Hi, my name is Laura Elias.

Today I'm gonna be showing you how to prepare the microinjection pipettes and load the pipettes for the int ventricular injections and electroporation that we will be doing. My name is David Castaneda and today I will be assisting with in vivo in utero injections of mice embryos. We will be injecting plasmid DNA into the lateral ventricle of developing mice embryos.

We target the CE cortex of these embryos so that we get expression of different molecules by glial cells and which will become Neurons later in development. When doing these intrauterine injections, there's a few important tools that you'll need to use and I'll show them to you.Now. We use a freer, a couple of addin forceps.

One, one pair that's serrated, and another pair that has a little tooth on the end. We use a pair of hemostats. This is a pair of scissors.

And then we use these two staplers. This is the larger ones that, that we use for rats. This is the smaller one that we use for mice.

Uses a size seven staple, and this is uses a size nine staple and they're just spring loaded. Some of the things that we use here to assist with the surgeries, we just use a simple fiber optic light source with a double head on it. We use a heated pad that just is circulates warm wa heated water through there to keep it warm.

The working surface, we use this por electroporation system made by BTX. We have a couple different sizes of paddles that we use based on how big, how large the embryos are. The blue side is the positive Side.

Okay, so For the mice we use an iso fluorine vaporizer. We found that it increases our survival rate of the mother and of the embryos. It it also allows us to bring the animal in and out of consciousness very quickly and in a very controlled manner.

So for that reason, we found it very useful. All right, so now we're going to bevel our injection pipettes. And this is very important so that your, your pipette is, has a large enough opening so that you can load your DNA, but also that it's very, very sharp so that it doesn't damage the embryo when you inject.

And if you damage the embryo when you inject, you're, you're gonna have a much higher rate of mortality. And so that's, that's not good. So this is actually a very important process as in this surgery.

And first we're gonna just put some water onto this beveling stone that's rotating here. And then we're going to take, let's see here, just a, a, a stick. And we're going to place our pipette that we just pulled onto this stick with some tape.

And we're gonna use one of these micro manipulators to hold the pipette on the bevel and allow it to bevel here. So, and just place this and we use microscope in order to visualize the tip of the pipette. And we'll just place it right down in the water on top of the bevel so that there's a little bit of pressure.

And you can see the tip pressing against the, the stone here. And allow it to bevel for some time for at least, you know, 15 minutes. I like to leave them even for, you know, an hour.

Sometimes they're just, they get really, really sharp, but it definitely depends person by person how long they bevel their pipettes. All right, so here we have, you can see on the top we have a pipette that's been pulled that hasn't been beveled yet. So while it's sharp, it doesn't have any opening through which you could load your your DNA.

Now the bottom pipette has been beveled and you can see this nice angled opening Now that will allow you to load your DNA, but it still has a sharp tip so that you can inject without creating any damage. All right, so now we're gonna fill our injection pipettes with DNA. So it's important that you prepare your DNA in an endotoxin free manner so that you don't inject any of the lipopolysaccharides when you inject your DNA.

So we're gonna take our DNA and actually here, get our paraform ready. So you're gonna just make your paraform here and you're gonna wanna collect your DNA and just place it as a little dot on this para film. And then we're gonna take, so I usually take for every 10 microliters of my DNA, which I usually use at about 1.5 to 1.7 micrograms from a microliter.

I'll use about one microliter of this fast green dye. And this is just to help us localize our injection and make sure that we've successfully injected into the ventricle. So then I'll just mix this fast green dye with my DNA.

And then you take your injection electrode and we'll just be filling the pipette by pulling back on the plunger. And you can see here if it, if the pipette is beveled correctly and has a big of enough opening, the DNI should load pretty easily. Sometimes you need to just move the plunger back and forth to help it load up correctly.

And so then you can load the pipette like this. And so we have the DNA loaded fully into this pipette and It's ready for injection. Okay, so I'm gonna wash the animal now with alcohol and iodine, but first of course I wanna do a toe pinch to make sure that the animal is fully under and it looks good.

So I can go ahead and begin. And I start out with just like in the rats, we do a series of three of these start out with the ethanol. Okay, Iodine.

To minimize the risk of infection, please notice that when William opened the wipes, he cleans the animal with the part that hasn't been touched by his fingers. So that to maximize that the area is clean. Second, he does only one wipe to avoid distributing germs over the abdominal surface.

Okay, great. Now I'm gonna clean up a little bit and we'll put on some surgical gloves.Okay.Okay.Okay. So now I'm going to open up the mouse and take out the uterine horns and I'll start that off by a little skin pinch.

Let me do another toe pinch here just to be sure she's fully under and that's good. So I'll pinch the skin and make just a little ripple there that'll stick up, which makes it easier for me to snip. And again, we want to make the incision right along the midline of the animal to avoid any blood vessels.

I usually cut the skin like that and I separate it and expose the muscle. And once you've done that, you can easily see the midline there in the muscle. It's very defined.

I'm gonna grab some muscle there snip And that's a good size incision. I wouldn't really want to go any larger than that. And then I use this freer just to lift up the muscle and the skin, pull it to the side, and then I do a very gentle probe in here and try and position the freer in between the two embryos in, in between two embryos so that I can just gently kind of tug them out without damaging them at all.

Sometimes it's easier than others. There we go. So just gently kind of pull it through and once I get it through, then I can just kind of nudge them out.

It's important never to pinch the embryos ever, but you just want to guide them, just kind of nudge them. And once you get them out, I wanna be sure to keep them always very wet. You don't wanna allow them to desiccate.

And let's see, I use my freer again here, probe around. There we go. That's great.

And so I always wanna make sure that I have them all out cause it's easy to leave one in there if you don't check for the ovaries. So I always pull them out very gently again, but looking for and looking to see the ovaries at the end, which is right there. And it just looks like a little mass of red tissue in this animal.

And then flip them over, come back and check the other side and I can see them there. So just be sure to keep them hydrated And we're ready to go. Something I want to point out is that You don't, you should grab the embryos very gently and only grab the embryos.

Don't grab where the blood vessels are because they will become, they can become damaged very easily and they will have, the animal will have a hemorrhage and the survival rate will drop dramatically. So the first thing that we want to determine is where the head is in the embryo that we want to inject with the DNA. We do that by rotating the animal gently and looking at the ventral, each ventral part.

So in this particular example we have that the head is on on to my right and the tail is close to my left. The midline is in this position and William is going to proceed to position the embryo in up appropriate way so I can inject it Just like in the rats. I always position them with the head on, the mother's right side facing towards me.

And so I'll start that now. Okay, so you can see the left floor pole there, move just a little bit and you can definitely see the midline right there. I'll try and spin it up a little bit so it's in good position for David.

Okay, And now I'm going to proceed to inject the left ventricle. We can see here that the left ventricle is totally, completely filled with DNA. We can see that some die has spread to the right ventricle and also to the third and four ventricles here.

That is a good indicator of good injection. So now we'll proceed to electro the DNA into the cells. The important consideration here is to know which electrode is the positive side.

We know that the one with the blue handle here is the positive electrode and that side should go in the side that we want to be electroporated the the DNA into. So since we have the, we injected the left ventricle with most of the DNAI will position the positive electrode on the left side like this, and then we will apply five pulses and that's it. It is important to have the electrodes completely wet in PBS so that the contact between the electrodes and the uterus is good and the transmission of the electrical pulses is Appropriate.

Okay, Now that the exploration is complete, I'm gonna go ahead and put the horns back inside. And how I'll do that is I'll very gently move the horns to one side, reach down in here and grab the muscle and the skin and kind of gently, gently easier back in there. Alright, Nice.

Be careful of the bladder. I can do the other side. And then what we do is just give her a little bit of a shake to kind of settle them down in there.

Can use the freer a little bit to flatten them out gently. And then what I'm gonna do is flush with three mils of antibiotic antimycotic. Let's do a toe pinch to make sure she's completely out before I start to suture.

And that looks good. I'm gonna use Henry Shine. Number six, surgical suture.

I prefer to do block stitches. I think they hold up better. And we're gonna start out just by putting the needle through there, pull it through, wrap around three times, grab the end, pull it through.

1, 2, 3, cut off the excess. So you want to just continue going down the line evenly space and pull tight. And you wanna go just beyond where the incision stops and tie off again.

And I'm approaching that now. Pull the tissue back or the skin back a little bit. There's a little bit of bleeding there.

It should be fine. We do keep a cauterizer around in case we get bleeding, which happens occasionally. And it's cauterizer works well to stop that.

So, okay, I'm beyond the incision. I'll stop there. Pull through, make a loop, spin around three times, grab the loop and pull it down.

Spin around three times, grab the loop, Pull it through, And turn off the excess. Put the needle in the sharps. And then we're going to use a stapler.

We're gonna be using size seven staples on this animal. We use seven for mice. Nine for rats.

Just grab the skin, pull it up a little ways, and start clipping. Grab the clip again. We want to go evenly spaced all the way down to a little bit beyond the incision and that's good.

Follow up with an alcohol wipe of the entire area. And then we're gonna give her an injection of buprenorphine just to make sure that she does an experience any pain. And we will continue to monitor her over the next couple days to make sure that she's staying healthy and not in any sort of pain.

Okay, so now that we've given her an injection of buprenorphine, we're gonna put her back into her original cage so she feels comfortable when she wakes up, put it into the incubator and allow her to stay in there for 20 minutes or so until she's fully awake and Dried off. We have Show you how to perform in vivo in utero, electroporation of plasma, DNA. We can inject any molecule.

It could be plaid D or it could be drugs. Growth factors dies. If you need these molecules delivered into the cells and they don't pass through passive diffusion, you will need electroporation and for that, molecules will have to be charged.

But basically the the possibilities are limitless. One example of an alternative vector for delivering genes into cell. Our viruses.

And in the past we have used retroviruses to label gls with DNA and show how they divide and become neurons, eventually migrating to the cortex after you label the cells with a virus or using electroporation, or you inject drugs or any other molecules to affect the development of the brain. You can study this by several techniques. One of them is do doing TimeLapse in which you will do organotypic slice cultures.

And then you will follow the fate of the cells using a a microscope. As you can see here, time 0.0. We start with a cluster of cells that are very close to the lateral ventricle.

And as time goes by, we can see that the cells start migrating out the ventricular zone and towards the cortical plate. In addition, we fix the embryos and we do immunohistochemistry to study, you know, different the distribution of different molecules, or to determine the fate of these different cells. Another possibility is to do electrophysiology.

We identify the label cells and then we study their electrical properties. Well, thanks for being with us today. We hope that you enjoy our demonstrations and that you will succeed in doing your experiments.

If you have any questions, feel, feel free to contact us. You know, good Luck.