Blairs are the embryonic cells formed during the cleavage stage of development. Here GFP labeled Blairs are transferred from one zebrafish embryo to another using a transplantation needle. This method allows the behavior and fate of blasphemers with altered genetic backgrounds to be followed in an otherwise normal embryo or vice versa by live microscopic imaging.
Hi, I'm Dr.Zoran, the laboratory of Dr.Shawe in the Department of Ophthalmology at the University of Pittsburgh. Today we'll show you a procedure for breast mial transportation in zebrafish embr. We use this procedure in our lab to study retina and the brain development.
So let's get started. In the evening, set up pairwise crosses of zebrafish to prevent random mating. Before the desired time, use dividers to separate the female and male fish.Here.
GFP expressing transgenic fish are used as a source of donor blastomeres. The PT 1 0 4 transgenic line expresses GFP ubiquitously under the control of the EF one promoter to prepare the agros wells that will hold the embryos pour melted 1%agros prepared in E three egg water into a Petri dish. Float a plastic mold with wedge-shaped protrusions on the agros solution.
Then let it cool down. To solidify carefully, remove the mold and immerse the agros bed in E three water. Next, prepare the transplantation needles.
Use a vertical needle puller at power setting 11.5 to pull glass capillary pipettes. Good needle tips should taper with long shafts. Grind the needle tips at a 45 degree angle with a micro grinder until beveled openings of 30 to 40 micrometers in inner diameter are obtained.
To clean up the needle tips, attach a hose to the blunt end, connect the hose to a syringe and then suck up and release a 10%hydrofluoric acid solution. Until all visible debris is removed. Wash the tips with distilled water.
Then with 100%ethanol, air dry the needles until all of the ethanol evaporates. It is important to ensure that the tips are completely dry. Residual ethanol will ruin the needles during the high temperature polishing.
Step two, to soften and polish the rough edges of the needle tips, bake them with the heat generated by the filament of a micro forger. Do not let the needle tips touch the filament while polishing the amount of electricity passing through the filament and the distance between the filament and the needle. Tips can be adjusted to attain desired heating conditions.
The temperature should not be too high. This will melt and deform the needle tips after the rough edges are smoothened. Carefully touch the heated filament with the needle tip and then gently pull the filament away to generate a pointed end.
A sharp, smooth and clean needle is critical for obtaining intact donor blasters without damaging the host embryos. Pull a glass pipette over a gas flame to make a fine glass probe with a smoothened end. This probe will be used to position the embryos in the proper orientation prior to transplantation.
On the day of the experiment, remove the dividers in the cross cassettes to allow fish to mate.Eight. After 30 minutes, collect 100 to 200 embryos by pouring them into a net. Place the embryos in a Petri dish filled with E three water.
Then using fine forceps, dec coate embryos by manually tearing away the CORs. Then use a glass pipette with a bent shaft to carefully transfer the coated embryos into aros wells. Do not let the embryos come in contact with air, which will cause them to burst.
Allow the embryos to develop at 28.5 degrees Celsius in the aros wells until three hours post fertilization or HPF while the embryos develop. Set up a transplantation apparatus by connecting the transplantation needle to a mineral oil filled micro pumping system. Make sure that the system does not leak and does not have any trapped air bubbles.
Backfill the entire transplantation needle with mineral oil except for the 0.5 to one microliter of space at the tip opening. Retrieve the embryos from the incubator and place them on the stage of a microscope to transplant blaspheme. Use a glass positioning probe to orient the blaspheme side of the three to four HPF embryos upwards.
Then gently insert the transplantation needle into a donor embryo and slowly suck up five to 20 blaspheme into the needle, making sure that the needle tip does not contact air during transplantation. Insert the blaspheme filled needle into a host embryo and release the blaspheme slowly. The site at which the blasphemers are released will influence where their progeny localize at later developmental stages to analyze retinal and brain development release cells at the animal pole.
If the development of other tissues and organs are to be analyzed, different releasing locations shall be considered accordingly. To minimize mechanical damage during transplantation, host embryos are left in the agros wells at 28.5 degrees Celsius until the next day. Then transfer the mosaic embryos to 24 well plates pre-coated with 0.5 milliliters of 1%aros and filled with one milliliter of E three egg water.
One embryo should be added to each well to prevent bacterial infections at 10 microliters of 100 x penicillin and streptomycin. To each, well weigh out and prepare low melting point or LMP aros and keep the solution in a 30 degree Celsius water bath. Transfer a mosaic embryo to a flora dish with a 0.17 millimeter thick glass bottom.
Remove any excess E three water to leave the embryo just covered. Then at 30 to 50 microliters of melted 1.5%LMP to the embryo quickly before the ARO solution solidifies. Position the eye or the brain as close to the glass bottom as possible.
After the solution cools down, add additional agros solution to further secure the embryos in place. Immerse the solidified agros block in two to three milliliters of E three egg water for confocal microscopy. A 40 x water immersion inverted objective with large focus depth is used.
Add a drop of water onto the objective and place a flora dish tissue culture dish on the stage and focus for image development. Perform time-lapse imaging by taking pictures of the embryos every five minutes. Depending on the nature of experiments, the time intervals between shots can be adjusted.
Here a representative image of a 24 HPF seber fish is shown. GFP expression highlights the donor cells in green. The retinal neuro epithelial cells span the entire thickness of the retinal wall.
This movie shows the movements of donor cells in the host brain at 24 HPF. Notice the cell morphologies change over time. Just show you how to perform breast mirror transportation in zebrafish.
When doing this procedure, it is important to remember to prepare a good needle on the transplant breast area gently. So that's it. Thank you for watching and good luck with your experiment.
