To begin lens transplantation in zebrafish GFP expressing transgenic donors and wild tap recipient hosts are first incubated for 30 minutes in separate pet dishes containing calcium free zebrafish ringer solution. Fish larvae are then embedded in two parallel lines in low melting point agros. One line A is for donors and the other B.For hosts, the entire donor eye is removed while only the lens is removed from the host.
The tissues surrounding the donor lens are removed before placing the lens in the host eye. After transplantation, fis are stored in labeled 24 well plates. The transplanted lens should dis display GFP fluorescence in the host.
Naturally, the contralateral eye of the host has not been operated upon and does not express GFP. Hi, I am Yj.And I'm Kaya McCullough, and we're from the laboratory of Dr.Yma Maki in the Department of Ophthalmology at Harvard Medical School. Today we will show you a procedure for lens transplantation in wild type zebrafish Daniel Rio.
We use this procedure in our laboratory to transplant lenses from wild type to mutant animals and vice versa. This allows us to determine whether mutant genes function in the lens autonomously or non autonomously. So let's get started.
Zebrafish strains are maintained in standard fish facility conditions at 28.5 degrees C on a 14 hour light, 10 hour dark cycle on the evening before the planned experiment plays males and females in a tank with a divider to separate them from each other. The genotype of these fish should yield progeny with GFP expression and any mutation of interest. In parallel use the same procedure to set up crosses between nont transgenic wild type animals within one hour.
After the light turns on in the morning, remove the dividers to allow the fish to mate. After 15 to 30 minutes, pour the tank water into a strainer to collect the embryos and then wash them out with a squirt of egg water from a wash bottle into a hundred by 15 millimeter Petri dish. Then clean the eggs by pipetting out detto on fertilized embryos and any debris.
Replace the old egg water with new egg water. The embryos can be stored in a 28.5 degree C incubator prior to the lens transplantation. When you're ready to proceed with the transplantation, prepare the embryos by removing the corion manually.
With a metal probe and a pair of forceps, the dec coated embryos must now be incubated in 0.2%EDTA in calcium free ZFR For 30 minutes. During the incubation, the needles required for the dissection can be prepared. The transplantation procedure requires a sharpened needle to cut tissues surrounding the lens and Woolies embryos from agros.
It also requires a blunt needle to move the donor lens and insert it into the host to make either needle first cut off the tip of a pasture pipet using a diamond knife. Then insert a glass capillary into the pasture piper tip so that about half of its length is inside it. Carefully insert a thin tungsten wire into the open end of the capillary using a flame.
Melt the glass over the wire with forceps steadily hold the glass end with a metal wire while twisting the other end of the needle. The softened glass should spiral around the metal, gripping it in place. This makes a blunt needle to create a sharpened needle.
Hold the tungsten wire over a flame for one to 1.5 minutes burning off the metal. So a very fine tip is created. Check the quality of the tip by microscopy.
A good tip should not bend or curve and it should taper smoothly to a fine point, no larger than 50 micrometers in diameter. Sterilize both needles in the flame for a few seconds and allow them to cool before beginning the procedure in a plastic centrifuge tube, prepare 1.2%low melting point aros and calcium free ZFR preheated to 40 degree C in a water bath at the desired stage, take up the donor embryos in a pasture pipet and slowly expel them into the centrifuge tube containing aros. Incubate the eggs in the aros for about five seconds.
Remove the embryos with the pipette and arrange them in a row on a hundred millimeter poly styrene Petri dish by pipetting slowly and carefully, most embryos will lie on their sides with the eye facing up. Now repeat this process with the host eggs and arrange them in a row parallel to the donor eggs before the agros solidifies. Use the blunt needle to turn any improperly oriented embryos so that the eye is facing up when the aros has solidified.
Overlay it with 0.2%low melting point aro solution. Using a sharpened needle, remove any aros around the eyes and very carefully cut out the entire donor eye, which will float.Next. Release the lens from the eye.
This is done by dragging the needle through the eye tissue from the bottom to the top. For the host only. The lens is cut out using small strokes with a sharpened needle, cut as close as possible to the lens without tearing it.
Avoid removing too much tissue from the rest of the host eye. Once free lenses will float in the medium. Using the blunt needle, carefully push the donor lens to just above the location of the host lens and then push it down into position with a blunt needle.
The host lens may be discarded. Leave donor and host embryos in 1.2%Aros for 30 to 60 minutes. Embryo medium will help wound healing.
Then release them from agros by using the sharpened metal needle. Transfer the embryos into a 24 well plate with embryo medium. Each embryo should occupy a separate.Well.
Make sure to label the well's properly so that it is clear which host corresponds to which donor after lens transfer is completed. Allow embryos to develop a 28.5 degree C until the desired time. For phenotypic observation, we usually examine lens differentiation within one to two days.
The donor derived lens should maintain GFP expression after transplantation into the host eye. If a mutant phenotype is not rescued in the wild type environment, this indicates that the phenotype is autonomous. However, if it is rescued, then the phenotype is deemed non-autonomous.
And this has important implications on the function of the underlying gene. We've just shown you how to perform transplant of the lens in the zebrafish embryo. When doing this procedure, it's important to be very careful with how you shape the needles and how you dissect out the lens itself.
Also, the embryos should be arranged quickly before the egg rolls happens once transplantation is complete. To not forget to add embryo, medium to eight in wood healing. So that's it.
Thanks for watching and good luck with your experiments.
