My name is Kelly Shea and I am the laboratory manager and also I'm a research technician in Neilsen's Laboratory, it's so difficult Because many people do not know how to distinguish the different tissue layers. So what you have to do is essentially peel back all of the tissue layers to expose the underlying embryo, which at this point is not very vascularized. So it's very transparent And it's hard to distinguish.
Take pretty blunt forceps and scissors. First thing you want to do is pinch the animal right around the midline and go ahead and make a first cut it. And then from that point you can just pull the skin apart, exposing the abdomen of the animal.
There's a thin layer of the peritoneum that you're going to pull up with the forceps, and then you can cut apart with scissors and just flap that back. And you want to cut along the meum of the animal and pull apart both uterine horns, which are connected at the middle. You can cut right at the base.
You're gonna probably get a lot of fat along with it. And take the entire uterine horn of the animal. Just place it right into the PBS.
So this is the in the horn. These are the implantation sites, 1, 2, 3, 4, 5. What you want to do is just separate them.
You can basically cut between the implantation sites right here. You can just grasp the embryo on its side, basically pry it apart. At this point, you're exposing the deum, which is a more spongy tissue underneath.
So the embryo should be Right in the middle, right here. This is the apex of the deciduous. You wanna orient it facing up with the apex.
What you're gonna end up doing is snipping off about one fifth of the way down from the apex. The embryo should sit right in the middle right here. What I end up doing is orienting it sideways, taking one pair of forceps and kind of grasping it, holding it down with the other, cutting off the tip of it, and then separating it.
So once that cap is removed, stand the embryo on its edge. And as you can see, you can, there's an opening right at the, where you remove the cap. Just want to pry the forceps in there.Yep.
So at this point, the embryo sits very loosely in in the deum. Once you pry the deum open, you can basically pop it out relatively easy as long as you get up underneath the base of it. So it's very tiny.
There are also some tissues still attached to the embryo. You have the ecto placental cone, which is right here at the apex of the embryo. And also you have some other tissues still attached.
So I just take pipette and suck him up. At this point, you want to fix the embryos. So I have just a dish of PFA.
We wanted to look Into candidate genes. Basically we're using whole mount RNA and situ techniques to visualize the production of nucleic acids, situ you within an embryo. These specific genes we're very interested in in code putative germ cell precursor markers, and also they're predicted to be expressed in the mouse embryo at a specific time point that helps the germ cells develop and mature from putative germ cell precursors into germ cells.
One such factor that we are very interested in first and, and the first factor that we actually did whole mount in situ on these day 6.5 embryos, was the bone morphogenic protein for BMP four. It's a member of the TGF-beta Super family. It's very important and is absolutely required in germ cell maturation.
And it's also required for the germ cells to translocate throughout the embryo and reside located near to the primitive streak of the embryo. And BMP four plays a critical role in both its signaling and the maturation that the germ cells need to go through in order to reside In the primitive streak. So you want to, number one, Make sure you know what you're looking for, you know how small it is, what scale you're working with, and also to sit down and plan very carefully.
The technique, especially with what kind of dissecting microscope you're going to be using. One of the technical difficulties is this involves a lot of manipulation and very, very steady hand movements so that you don't damage the embryo. So you want a dissecting microscope where you have a lot of room to manipulate, and also make sure that you have everything set up that you need for the experiment so that you're not doing much more manipulation than what you need to be doing within the dissection.
So other than that, I think you need to take it very slow and peel back the individual tissue layers very slow so that you know what you're working with. And make sure you know where the embryo sits.
