移植によるキメラゼブラフィッシュ胚の生成

Hi, I'm Hillary Kemp from the Laboratory of Cecilia Owens. And I'm Cecilia Owens. Hillary and I are in the division of basic science at the Fred Hutchinson Cancer Research Center in Seattle.

Today we're going to be showing you a technique for making chimeric zebrafish embryos, also known as genetic mosaics. This procedure allows us to assess the cellular basis of gene function during development. We can ask whether a given gene functions within the cells with a mutant phenotype known as cell autonomous function, or within those cells environment known as cell non autonomy.

We make these mosaics by transplanting cells between wild type of mutant embryos at either a blastula or early gastro stage. We subsequently assess gene function by observing the phenotype and genotype of donor derived cells in their host environment. So let's get started.

In this part of the protocol, a lineage dye is injected into the donor embryo so that the fates of donor derived cells can be followed after transplantation. Any variety of nucleic acids can also be injected using this technique, however, omit the decoration step when injecting DNA before working with the embryos, prepare an injection dish by inserting a glass slide at a 45 degree angle across the widest part of a Petri dish. Three quarters full of 1.2%aros in pen strep embryo medium.

Remove the slide after the agro sets to make a beveled trough. Fill the trough with pen strep embryo medium. Now proceed with the enzymatic decoration of one cell stage embryos.

Incubate the embryos at the one cell stage for one to five minutes in a 0.5 milligram per milliliter. Pronase solution. Monitor the decoration closely.

As soon as the corion begin to visibly collapse, submerge the embryos in system water And this is pouring off all the corium and the embryos are staying down behind at the bottom of the beak curve. And it's really important that the embryos don't hit the surface of the water just one more time, and most of them have their corion off. There's a few swirling around the outside that don't.

If anything still is in its corion, then the act of pipetting them like this will get them out and then release the embryos so that they never hit the surface of the water. It is important to handle coated embryos with a pipette that has been fire polished to smooth its edges. Gently transfer the fragile dec coated embryos into the trough of the injection dish using a wide bore fire polished glass pipette.

And then we're loading a single file into this trough. Now the embryos are ready to be injected with lineage marker using a precisely calibrated fire, pulled glass injection pipette, and a pressure injection rig. We're going to use a dish full of embryo medium to break the needle and a dish full of oil to calibrate the needle.

This is just a regular standard lab grade mineral oil. We take a pair of sharpened watchmakers forceps and then while looking through the eye pieces to determine what the right size would be, we put the forceps on either side of the needle and then we gently break the chip off, which I can't actually do without looking To determine the volume of dye or ribonucleotide being delivered. Before starting.

Measure the bolus size in a dish of mineral oil. The needle should be broken off so that a bolus of one nanoliter is delivered with one or two pulses of the micro injector. The volume of the bolus can be calculated from its diameter as measured by an eyepiece micrometer.

Inject one nanoliter of one to 3%solution of fluorescent dextran directly into the ylk of coated embryos between the one and four cells stages. What I'm going to do is position the embryo right under the needle. I'm gonna jab the needle gently into the center of the yolk and depress the foot pedal.

Once injecting, It is important to inject into the yolk streams so that the dye is transported into the cells. This injection will be sufficient to track cells in chimeric embryos through several days of development. Ideally, we do this before the cells reach the four cell stage, Raise the injected embryos at low density in an coated Petri dish filled with fresh pen strep embryo.Medium.

Note that injected embryos tend to have a slightly slowed rate of development, incubate embryos at different temperatures, 25, 28, and 31 degrees Celsius to stagger their development and maximize the time window over which transplants can be performed. The apparatus used for cell transplantation in the zebra fish blastula consists of a drive controlled Hamilton syringe attached by a three-way stop cock to a reservoir of mineral oil and to a micro pipette holder through a length of flexible tubing. Eliminate all air bubbles from the system as they will ruin your ability to control the suction and pressure.

Use a stereo microscope that has high quality optics of at least a DX magnification. To control the positioning of the micro pipette holder and needle, we suggest a NARA shige manual micro manipulator. Be sure it is fixed to the bench using a magnetic base to prevent vibrations from transferring to the transplant.

Needle transplantation needles are made from glass capillary pipettes that are drawn to a gentle taper on an electrode polar. See the JoVE protocol from the lab of Miriam Goodman for details on pulling pipettes under a dissecting microscope, break the tip of the pulled needle off. For blastula stage transplants, the outer diameter of the needle should measure approximately 50 to 60 microns or 30 to 40 microns for a gastro stage transfer.

For a gastro stage transfer to improve penetration, a sharp barb needs to be pulled on the end of the needle using a micro forage first, smooth the tip of the needle by bringing it close to the filament of the micro forge. Then turn the needle so that the leading edge of the bevel can make contact with the filament. Once the leading edge of the needle contacts the filament, pull it away quickly to create a barb.

To avoid damaging cells, the barb should be straight and the tip of the needle should not curve With the rig and transplantation needles prepared, you are ready to make chimeric embryos. Blastula transplants are used to transplant cells between embryos when fine fate map information is not required. With the exception of primordial germ cells, most cells are generally not committed to a fate at this stage.

The fate map of the blastula is such that we can distinguish between cells that are gonna give rise to ectoderm and cells that are gonna give rise to mesoderm. The mesodermal and endodermal lie near the margin of the blastula stage embryo, whereas dermal progenitors, which are going to give rise to the nervous system and to the surface derm, lie further away from the margin of the embryo. So when we transplant cells into blastula stage embryos, we can target those cells to the presumptive mesoderm or to the presumptive ectoderm.

But we can't target cells to any particular region of the ectoderm or of the mesoderm because we don't know. At the blastula stage where the dorsal side of the embryo is, Prepare injection dishes in advance by floating a plastic mold with paired rows of wedge-shaped protrusions in a Petri dish, half filled with molten 1.2%aros in embryo medium. Once the aros has solidified, remove the template leaving rows of triangular shaped wells each just large enough to hold one embryo.

Fill the transplantation mold with pen strep embryo medium, and load blastula stage embryos individually into each well with a fire polished glass pipette. Now transfer the embryos to the transplant wells initially allow them to lie on their side. Arrange the embryos so the donor embryos are placed down one column and the host embryos are placed down the adjacent column.

Position the dish on the stage so that when the transplantation needle enters an embryo, the needle pushes the embryo against the back wall of its well. Using the micro manipulator, lower the transplantation needle into the dish at a fairly steep angle. Ideally, the transplantation needle should enter the embryo at approximately a 45 degree angle.

Once the tip of the needle is below the surface of the embryo medium, draw a small amount of embryo medium into the needle by twisting the micrometer drive controlling the Hamilton syringe. For the most precise control, the interface between the mineral oil and the embryo medium should remain in the thin tapered part of the needle, not too close to the end. Gently position the donor embryo with the needle and then draw the needle back and enter the blastula cap of the embryo at the desired position.

A swift hard hit will penetrate the embryo without causing it to roll. Draw up donor cells slowly and carefully into the needle to avoid shearing the cells. Avoid taking yolk up into the needle after the desired number of cells is taken up.

Reverse the pressure slightly to stop the suction and remove the needle from the bring the host embryo into position. By moving the transplantation dish, small adjustments to the position of the host embryo can be made by gently turning it using the transplantation needle as long as care is taken to not touch the yolk. When expelling the donor cells into the host embryo, avoid taking ylk up into the embryo as this will damage and kill the donor cells from a single donor embryo can be transplanted to multiple hosts after the cell transfers have been completed.

Transfer the donor host pairs to the coated wells of a 24 well plate to continue developing. The one exception to the rule of cells not being committed to particular fates in the blastula stage is the primordial germ cells. Primordial germ cell cells are specified very early in development and are committed to their primordial germ cell state fate.

From a very early stage, the primordial germ cells lie near the margin of a blastula and a gast stage embryo. So if the goal of the transplantation experiment is to transplant primordial germ cells, then it's very important where the cells come from in the donor embryo and not important where they go to into the, in the host embryo. So the rule is that for somatic cells, it's important where they go to in the host embryo that will determine their fate.

Where in the case of primordial germ cells, it's important where they come from in the donor embryo because their fate is already determined. If the goal is to transplant primordial germ cells, then this, the cells need to be picked up from the margin of the hope of the donor embryo, and they are situated right near the margin of the embryo in four little clusters of each of three or four cells at this sheet, this spear stage or high stage of development. And you have to pick them up very gingerly so as not to accidentally start sucking up the yolk.

Okay, and so that's what I've done there, and now I'm moving to a host embryo. Now the primordial germ cells will hone to into the genital ridge region of whatever embryo they're in. So there's no need to transplant these primordial germ cells to the margin they'll get there themselves.

So I'm just transplanting putting them back in any old place into the post embryo. The key for germ cell transplants, which is quite the opposite of any other kind of transplant, is that it matters where the donor cells come from. Transplanting cells into a gastro stage host allows the experimenter to take advantage of the zebra zebrafish fate map that emerges when the embryonic shield becomes visible.

It becomes possible to distinguish different tissue types as well as the domains within the presumptive central nervous system. When transferring cells between embryos at the gastro list stage, there are some variations in the protocol, but the general procedure is similar to working with blastos to mount gastros. First smear a vertical stripe of Methylcellulose in the depression of a glass depression slide, then flood with a generous amount of pen strep embryo media using a fire polished pipette transfer.

One donor and three shield stage hosts to the depression slide. Select donor embryos that are slightly younger than the hosts. A new tool is required to manipulate the gastros glue, the ends of a short length of two pound test fishing line into the end of a capillary tube.

Thus making a small loop. Use the loop to gently roll the donor and host embryos up onto the top of the Methylcellulose strip and stuff them down into the Methylcellulose until secure. Roll them into position so that the targeted region is uppermost.

Since during transplantation, the needle will enter the embryo tangentially to deliver the donor cells without damaging the ylk. Gastro stage transplants are best performed on a fixed stage compound microscope. Use the controls of the micro manipulator to bring the tip of the transplantation needle into focus.

Position the micro pipette holder and needle at a fairly shallow angle so that the needle is as close to horizontal as the edge of the depression on the slide will allow. Next pierce the donor embryo. If it is mounted properly, it will not roll as the transplantation needle enters unless the embryo is too old to allow the needle to penetrate easily.

To avoid piercing the ylk, the needle should enter the embryo at a focal plane that is just slightly deeper than that of the EDL. Once pierced, a large number of cells can be removed from the donor for up to three host cell injections. When drawing up cells, avoid sucking up the yolk by moving the needle around as the cells are taken up if desired.

Additional embryos can be harvested for more cells using the same needle before ejecting the cells into the host, during cell ejection into the host embryo expel the cells while the needle is being drawn through the targeted area as opposed to depositing the cells in a single clump. This will increase the likelihood that the donor cells will contribute to the desired tissue. Once the cell transfers are completed, place the entire slide into a Petri dish and carefully flood it with pen strep embryo.Medium.

Over the next few hours, the methylcellulose will dissolve releasing the embryos. The number of cells transferred depends on the specific objective of the experiment. Cells from a single donor can be transplanted into three or four hosts the morning after transplantation.

Hosts can be examined to determine if donor cell targeting was successful. Successful transplantation of primordial germ cells from blastula age results in fluorescently labeled cells, ventrally positioned near the junction between the ylk and the ylk extension. So we've just shown you how to do gastrula and blastula stage transplants.

When doing this technique, it's important to choose the right type of transplantation method for the target tissue that you're hoping to target with your cells. So blastula stage transplant is sufficient for determining the cells go to the derm versus the ectoderm. And you can also use this technique to target primordial germ cells.

If you would like to do finer mapping, it's better to use a gastros age transplant where the dorsal side has already been determined. And so with this technique, you can target cells to various parts of the nervous system, in particular, the forebrain, midbrain, hind brain and spinal cord. So that's it.

Thanks so much for watching and best of luck with all your experiments.