組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

This procedure begins by lining single cell zebrafish embryos into a microinjection plate. Micro inject a fluorescent dextrin solution into the yolk. Just below the cell injected embryos are your transplant donors.

Coate donor and host embryos and grow to the shield Stage load two DEC coated dextrin injected embryos side by side into one well of a transplant plate. Repeat below each donor pair. Add one host embryo per well to target transplanted cells to the future di cephalon of the forebrain transplant cells from and to the midline.

Halfway between the animal pole and the shield. Transplant cells from the donor gasa into the host Gasa. Grow to 30 hours fix.

Perform immunochemistry if necessary, mount and image. Alternatively, mount the host live and conduct time lapse imaging. Hi, my name is Elizabeth De Shane from the laboratory of Dr.Michael ESI in the Department of Biological Sciences at Smith College in Northampton, Massachusetts.

And I'm the principal investigator of the laboratory, Dr.Michael Besi. Today we will show you how to create chimeric embryos by conducting gastric stage cell transplantations using the zebrafish model system. We use this procedure in my laboratory to study the function of specific genes on the development of forebrain commissure as well as the process of axon glial interactions during commissure formation.

So let's get started. To begin prepare trough molds for the microinjection plates by carefully breaking three one millimeter, four inch long non filament capillaries in half. Using super glue, glue two of the capillary halves side by side onto a flat surface glue.

The third capillary on top of these two nestled in the groove to create a pyramid shape. With the three capillaries, allow the mold to dry. You should make around three to five molds for each injection plate.

Next, prepare the injection plates by pouring 20 to 25 milliliters of molten 1.5%aros in embryo medium into a 100 millimeter Petri dish. Immediately place three to five of the prepared trough molds into the bottom of the plate, ensuring that the flat side of the mold is touching the bottom of the Petri dish and is completely covered by aros. When the aros has solidified, remove the trough molds by cutting out a large aros square that contains them.

Gently dislodge the agar on the periphery of the cut square and discard it. Flip the square agar piece over and using fine forceps completely, remove the trough molds. Pour molten aros into the Petri dish around the square to hold the newly molded wells in place.

Let it set. Plates can be paraform sealed and stored for weeks at four degrees Celsius. They should be discarded if there are any signs of growth in the agar.

Finally, to prepare the injection needles use a micro pipette capillary puller to make the needles out of one millimeter four inch glass capillaries with internal filaments with the micro injection plate and needles prepared. Let's look at collecting embryos and setting up the microinjection apparatus. Fertilized eggs are collected immediately after they are laid and maintained in a Petri dish filled with embryo medium.

Using a wide tipped glass pipette expel embryos into the wells of a room. Temperature injection plate filled with embryo medium. Use clasped blunt forceps to gently wedge the embryos into the bottom of the troughs embryos whose corian or yolk have been compromised should be discarded.

Next, load the injection needle with one to two microliters of diluted fluorescent Alexa 5 9 4 dextrin solution. Allow the dextrin to travel to the tip of the injection needle by capillary action. Attach the injection needle to the capillary holder of a stereotactic micro manipulator and adjust the pressure on the micro injector apparatus to begin with.

40 to 50 PSI and a pulse duration of 500 milliseconds. Use forceps to gently graze or snip the tip of the injection needle to break the seal. Ideally, the diameter should be 0.05 to 0.15 millimeters.

To calibrate the needle, use a stereo microscope and expel the dextrin onto a micrometer with mineral oil on top of it. Modify the bolus size by adjusting the air pressure duration of the pulse and the size of the needle.Tip. A bolus size equivalent to a volume of 0.8 to one nanoliters should be maintained throughout injections.

We can now begin microinjection of dextrin using about 3.2 x magnification on a stereo microscope. Start embryo injections from one end of the trough by smoothly piercing the corion and cell membrane of embryos to enter into the yolk. Inject the dextrin solution just under the cell.

Make sure to remove the injection needle with the same smooth movement used to enter the embryo. Move the Petri dish in order to position the next embryo in line and continue injecting down the length of the trough. Check the pressure and bolus size periodically.

Now use forceps to gently push the embryos out of the troughs. Transfer them to a Petri dish filled with antibiotic embryo.Medium. Incubate the embryos at 28.5 degrees Celsius and monitor them for unfertilized eggs or death.

The embryos are now ready for future transplantations. Prior to the completion of a poli during gas ration, portions of the yolk remain exposed. The yolk is extremely fragile and will adhere and easily tear on the plastic of the Petri dish.

Therefore, embryos need to be coated on a coated plates a day before the transplants take place. Make these dec decoration plates by pouring five to 10 milliliters of molten aros into a 100 millimeter Petri dish. Only a thin layer of aros about two to three millimeters is needed to cushion the embryos during decoration.

Let the plates cool. Next, make separate transplantation plates by pouring 30 milliliters of 1.5%aros solution into a 100 millimeter Petri dish and insert a transplant well mold. The mold should contain 104 triangular divots, each comprised of three 90 degree sides and 1 45 degree angled side.

The wells are designed to hold two embryos side by side. Take care not to trap air bubbles under the mold and let the agros harden. Removing the mold reveals a sunken area containing the square wells with a sloped edge.

To prepare host and donor embryos for transplantation, their development needs to be monitored to maintain near age-matched pairings. To do this, embryos can be raised at varying temperatures from 23 to 31 degrees Celsius to slow down or speed up development. Both host and donor embryos should be hand coated on an coated coating plate one hour prior to the desired age.

In the case of gastro stage transplants, this would need to be completed by five hours post fertilization or HPF prior to the shield stage at 5.5 HPF. Use a glass pipette to load the host and a donor embryos into a transplant plate filled with antibiotic embryo medium. Here the first row is filled with donor embryos with two embryos per well, and the following two rows are filled with a single host embryo per well.

For transplants, we use a Zeiss luer fluorescent stereo microscope that has fully automated joystick controlled zoom and focus manipulation. While this automated microscope is not necessary, it does dramatically increase the ease and efficiency of these fine surgical transplants, especially when a high number of transplants are desired. To set up the transplant apparatus, set the einor air tram to the middle location of its scale, connect the air tram tubing to the capillary holder and secure the holder to the einor automated micro manipulator With the embryos and transplant apparatus prepared, we can now move on to gastro stage cell transplants.

The most critical skill in completing gastro stage cell transplants is the ability to recognize the cellular anatomy of the dorsal blasts APO lip, also known as the shield in zebrafish. Here we present a sequence movie of the gastrula from different viewpoints and highlight where the shield is located in this lateral view. The shield or dorsal side is to the right in the red outline to target cells to the forebrain transplant cells from and to the midline.

Exactly halfway between the animal pole and shield, we will begin rotating the embryo first towards you to view the animal pole, highlighting the shield and transplant site along the way. Next, the embryo spins on its axis to position the shield at the bottom. The gastro now rotates back up, presenting you with a straight on dorsal view.

The embryo is now rotated back in sequence to a lateral view at six HPF. Using the micro manipulator gently graze the embryo with the tip of the capillary to rotate it into a position for cell extraction such that its midline and shield are visible and oppose the tip of the capillary donor embryos are easily verified by switching from brightfield to fluorescent illumination. Here the cells of the gastro fluoresce red aspirate a small amount of embryo medium into the capillary to eliminate any chance of cells contacting the air water interface that would damage them.

Gently pierce the ectoderm of the donor embryo at the midline exactly between the animal pole and the shield. There was only approximately 20 micrometers of cells between the surface ectoderm and the underlying yolk in the gasa. So be careful not to impale the yolk as this will often lead to death within hours.

Using the air tram slowly aspirate cells into the capillary. Always maintain visibility of the water line slightly agitating the tip. While inside the donor will help to loosen cell to cell contacts.

Approximately 10 to 50 cells should be extracted from the donor embryo. Next, remove the capillary from the donor. Similarly orient the host and pierce and expel the cells into the exact same location In a host embryo, switching from brightfield to fluorescent illumination reveals the movement of the dextrin labeled cells in the capillary needle into the host embryo.

Finally, after transplantation, remove the donor and host embryos from the wells. Separate them and gently place them on an A coated Petri dish filled with antibiotic embryo. Medium incubate the embryos at 28.5 degrees Celsius with the transplants complete.

Let's begin visualizing and imaging. The embryos begin by fixing embryos at the appropriate stage for imaging in an immunochemistry verified preservative. In our case, this would be in 4%formaldehyde for two hours at room temperature or overnight at four degrees Celsius rotating.

Follow this by rinsing fixed embryos two times and then washing them for three to five minutes. In 0.1 molar pb. Perform proper immuno labeling if need be.

Place the embryos in 75%glycerol, sink them at room temperature and store them at four degrees Celsius. Prepare a slide to mount two embryos by creating two petroleum jelly square outlines on a slide that match the inside diameter of a square. Cover slip next de yoan embryo and perform any necessary dissections to achieve a frontal view of the forebrain.

Dissect off the forebrain by cutting perpendicularly across the midbrain. Place this tissue with a minimal amount of glycerol within the petroleum. Well orient the tissue into the appropriate position and place a cover slip over the specimen.

Alternatively, for live embryos, mount them in a 0.75%agro solution onto glass. Bottom culture dishes before the agro solidifies, use a tungsten needle to manipulate the embryo into the proper orientation for imaging. Both fixed and live hosts are imaged.

Using the Leica SP five laser scanning confocal microscope, ZS stacks are acquired and three or four dimensional image processing is conducted using velocity software to generate chimeric embryos in which some portion of the astro glial population within the embryo is different by genotype or phenotype. We used a multifaceted approach that combines the use of GFP transgenic embryos, which labels astroglia with the use of gastro staged cell transplantation to specifically target our clones to the cephalon. We utilized the zebrafish gastro fate map to selectively extract cells at the midline equidistant from the animal pole and the shield.

These extracted cells were then transplanted into the same location in a wild type host gasa, which was then raised to 30 HPF and immuno labeled for all axons as referenced landmarks fore brain anatomy. In the example shown here, confocal imaging of one host embryo reveals isolated GFP positive cells throughout the cephalon and cephalon. The full morphology of these GFP positive cells can be observed in this clonal assay revealing that most of these cells take on the characteristic radial glial morphology where the soma is located adjacent to the ventricular zone and a large end foot terminates a radial process at the peel surface.

Three dimensional rendering of the Zack of these collected optical slices clearly shows the position of these cells with respect to the labeled axons. Another approach commonly used for visualizing transplanted cells is to initially micro inject a fluorescent cell lineage dye into the yolk of a one cell staged wild type or GFP transgenic embryo. In this example, we injected Alexa 5 94 dextrin into wild type embryos.

At the one cell stage we allowed them to develop to shield stage gasa and then carried out four brain targeted transplantation as mentioned above. But instead, we transplanted into transgenic host gastrula imaging of the dorsal cephalon by laser scanning. Confocal microscopy revealed fluorescent red clusters of donor cells amongst GFP positive nuclei within the forebrain.

We further analyzed this host by collecting Zacks every three minutes over the course of two hours with the laser scanning confocal. Four dimensional rendering of this time lapse using velocity software by IMP provision shows dynamic cellular movements of the rod domine cell membranes and the GFP positive nuclei. We've just shown you how to prepare all the necessary apparatus and reagents for cell transplantations in zebrafish embryos.

Specifically, we demonstrated the technique at the gaso stage. However, it's also applicable to blastula stage transplants. When using this procedure, it's important to precisely correlate the final desired location of the transplanted cells with the earlier fate map location of these cells in the gas consult, Dr.Scott Frazier's detailed study of zebrafish fate maps from 1995 entitled Fate Maps of the Zebrafish Embryo for further guidance.

So that's it. Thanks for watching and good luck with your experiments.