The overall goal of this procedure is to produce clones in an embryo for the purpose of examining the autonomy of a gene or protein of interest. This is accomplished by labeling donor embryos with Rod Domine dextran by micro injection at the one cell stage as shown in the companion to this video, microinjection of Maka embryos. The second step of the procedure is to develop both donor and recipient embryos to the correct stage.
The third step of the procedure is to coate both the donor and recipient's embryos. The final step of the procedure is to transplant cells from the labeled donor embryos to the recipient's embryos. Ultimately, results can be obtained by analyzing the distribution proliferation and differentiation of clones, of transplanted cells through detecting transplanted cells by their fluorescence or lineage tracers.
Hi, I'm Sean Brazinski from the lab of Dr.Makoto Fki in the Department of Biology and Biochemistry at the University of Bath. I'm wan also from the Zeki lab. Today we'll show you a procedure for cell transplantation in the dark fish embryos.
We use this procedure in our lab to study with a gene or mutation of interest, has a cell or non cell autonomous function. So let's get started. When coating maka eggs use sterile solutions and tools to enhance the success of long-term observation and culture.
Place a piece of P 2000 grit waterproof sandpaper in the lid of a nine centimeter non-adherent Petri dish. Use a wide mouth to fire polished pasta pipette to transfer up to 10 freshly unclustered cleaned and labeled eggs from a dish of egg. Medium to the sandpaper.
Remove excess medium, leaving just enough in the dish to keep the eggs from drying out. Use a gloved fingertip to gently roll 10 eggs at a time over the sandpaper for 45 to 60 seconds. To remove outer surface hairs and score the Corian surface, apply minimal pressure and keep the fingertip parallel to the sandpaper.
Transfer the eggs to the original dish of medium. Remove the medium, cover the eggs with a solution of 20 milligrams per milliliter of prone. Cover the dish and incubate at 27 degrees Celsius for 60 minutes.
Being sure to place the dish at an angle so the embryos are sufficiently immersed in enzyme. In order to minimize enzyme required, use a plastic transfer pipet to recover pronase for reuse and place it on ice. Wash the eggs five times an embryo medium to remove all traces of prone to prevent it from digesting the hatching enzyme.
Add enough hatching enzyme to cover the washed eggs. Ensure the eggs remain in a single layer on the bottom of the dish to avoid crushing as the Corian dissolves. Incubate the eggs at 27 degrees Celsius.
Being sure to place the dish at an angle so the embryos are sufficiently immersed in enzyme. In order to minimize enzyme required check progress under a dissecting stereo microscope, create alike holes will appear in the inner layer of the Corian before it dissolves completely. The process may take up to 60 minutes depending on enzyme activity.
When a small part of the Corian is dissolved, immediately suck up the individual egg with a pipette to avoid digestion of the embryo by the hatching enzyme. Transfer to a clean dish of one times BSS by touching the tip of the pipette gently to a clean dish of one times BSS and letting the egg roll out to minimize transferring hatching enzyme. Use forceps to remove the rest of the Corian when all the eggs have been transferred from the hatching enzyme.
Make a final transfer to a new dish of one times BBSS. Add antibiotics if the embryos are to be brought to a later stage of embryonic development After decoration for this procedure, begin dec coating stage 12 embryos 1.5 hours before transplantation. Use a pipette tip to add a small amount of 3%methyl cellulose to the center of a cavity microscope.
Slide in a nine centimeter petri dish. Spread the liquid thinly over the surface of the depression. Dry the methyl cellulose for two minutes.
Fill the slide depression with 350 microliters of sterile one times BSS under a dissecting microscope. Use a wide mouthed fire polished glass pipette to transfer one donor and up to four recipient embryos to the slide. Use a hair loop to orient the embryos so that the blaster derm is facing upwards.
Lean the embryos against each other for stability if necessary. Gently insert a microneedle into the donor blaster.Derm. Slowly uptake 10 to 20 cells.
Now gently insert the needle into the relevant area of the recipient embryo blaster derm slowly expel the donor cells. Do not disturb the cello sac boundary. Carefully fill the Petri dish with approximately 30 milliliters of sterile one times BSS to immerse the embryos and loosen the embryos from the methyl cellulose.
Pour against the side of the dish so as not to disrupt the embryos. Add 100 microliters of penicillin streptomycin into the dish for an approximate concentration of 0.3%Cover the dish. Observe the embryos periodically as necessary.
For longer imaging studies such as time-lapse and detailed observations, embed live coated embryos in aros anesthetize live embryos to prevent movement by adding a stage appropriate anesthetic to the medium containing the embryos melt approximately one milliliter of 3%Low melting point arose in one times BSS and keep it at 37 degrees Celsius. If needed, add an appropriate amount of anesthetic to the aros to prevent twitching working quickly to prevent solidification, use a wide mouth pasta pipette to fill the cap. Depression of a micro centrifuge tube with aros.
Use a wide mouth pipette to move one coated and anesthetized embryo into the aros in the cap. Transfer as little medium as possible with the embryo. Immediately update the aros and embryo and move them into a 3.5 centimeter glass bottom Petri dish.
Transfer the dish to a 14 centimeter dish filled one third of the way with an ice and water mixture. Hold the smaller dish down firmly to the bottom of the larger dish under a dissecting stereo microscope. Use a hair loop to quickly orient the embryo as desired.
Hold the embryo still until the agro solidifies. The embryo can now be imaged. Image A shows a donor and recipient embryo immediately post transplantation.
The donor embryo is shown on the right with a completely red labeled plaster derm. The recipient embryo is shown on the left and is easily identified by the small mass of red transplanted cells visible in the blaster derm image B shows two recipient embryos approximately seven hours post transplantation. Notice the transplanted cells have migrated from the site of transplantation and are now dispersed across the blaster.
Derm image C shows a recipient's embryo at stage 29. Note the pigmentation in the eye and the presence of mefor in the trunk and brain region. The rod domine labeled cells can be seen to be colonizing many of the embryonic structures in indicating the successful production of a chimera.
We've just shown you how to ate meca fish embryos and carry it out cell transplantation at an early embryonic stage. When doing this procedure, it's important to remember to transplant your donor cells into the correct area of the recipient blasted inm. This will allow your donor cells to differentiate into the correct cell type.
So that's it. Thanks for watching and good luck with your experiments.
