The overall goal of the A CT assay is to test whether a gene or genes of interest can specify primitive derm toward a particular cell fate. The procedure starts by first over expressing the RNAs in coding of fluorescent reporter gene and the gene or genes of interest in developing XUS lavis embryos. The next step is to dissect the animal cap or primitive odum expressing the overexpressed RNAs and grow them to stage 15.
The third step of the procedure is to remove tissue on the flank of a stage 15 host embryo. The final step is to transplant half the donor animal cap tissue onto the flank of the host embryo and grow the host to stage 42 to 43. The results show ectopic tissue formation such as eye like structures through sectioning and immuno staining for tissue specific markers.
Hi, I'm and sine in the Department of Ophthalmology at Upstate Medical University. Today I'll show you procedure that we call the animal cap transplant assay that I developed in collaboration with Mike Suber. We use this assay in both of our laboratories to study the genes that are sufficient for eye formation.
So let's get started. To begin this procedure, prepare the cap RNAs for injection transcribe noggin or another gene of interest and YFP to be used as a tracer. Also prepare the MMR based solutions, a stock of eight x egg laying solution and the needles for injecting the embryos.
A custom made elastomer mold is used for making embryo holding dishes. Generate one injection plate per experimental condition by melting 1%AROS in 0.4 XMMR in a microwave. Transfer about six milliliters of the solution into a 60 millimeter plate.
Gently place an elastomer mold on top of the solution to prevent bubbles and let cool for about 20 minutes. At room temperature, generate one cap isolation plate per experimental condition, using 1%AGROS plus 0.7 XMMR with molds as was just shown for the injection plates. Prepare extra plates for each time point.
Consult the accompanying written protocol for details of calculating the time points. Next isolate. Test these from a male frog and store the tissue at four degrees Celsius in a sterile solution of one XMMR supplemented with 50 micrograms per milliliter of Gentamycin.
This can be done up to a week before the start of the experiment, but note that the sperm loses efficacy over time, late in the day on the day before microinjection. Inject females with 500 units of human chorionic gonadotropin. Place females at 16 to 18 degrees Celsius overnight.
Now all is ready for microinjection the following day. Start by removing the hormonally induced females and place them in individual tanks containing one XELS. Prepare the needle for microinjection by snipping off the tip.
To obtain a 25 to 35 micrometer diameter, place the needle onto the PLI 100 pico injector and calibrate it so that it dispenses 10 nanoliters per injection. Set an incubator to 14 degrees Celsius by placing the exit this lower temperature after injecting them with RNA, the development of the embryo is slowed down and many more transplants can be performed in one experiment about an hour before fertilization, remove the eggs from the tank. Once females have laid eggs for an additional hour, use a transfer pipette to remove the newly laid eggs from the tank.
Note not to use eggs that have been in the ELS solution for more than two hours. As the quality of the eggs is reduced, place the eggs into 60 millimeter petro dishes. The dish is about 80%full of a single layer of eggs.
Remove as much ELS from the eggs as possible and replace with one XMMR. The eggs can stay in one XMMR for up to an hour. Next in vitro fertilize the eggs with homogenized testes.
It is best to fertilize the embryos in three separate batches at around 11:00 AM noon and 1:00 PM so that they can grow into the desired stage. 8.5 to nine for cap isolation and stage 15 for transplantation. An hour after fertilizing the eggs, remove the jelly coat on the eggs using D jelly Solution Wash with copious amounts of 0.1 XMMR and then wait until the fertilized eggs reach the two cell stage.
To begin sorting. Prepare an injection dish filled with 0.4 XMMR plus 6%F ccal. Once the embryos reach the two cell stage, use a transfer pipette to sort uniformly pigmented and evenly dividing embryos into the well of the injection dish.
Proceed to Microinjection first stretch perfil over the top of a 60 millimeter petro dish lid. Pipet two microliters of the RNA sample onto the biofilm and aspirate. Using the pico injector to fill the injection needle.
Be sure to check that the needle is still dispensing 10 liters of sample before starting injection. The RNA is injected into both blasters over two cell stage embryo. Inject 20 to 30 embryos each with 500 picograms of Y-F-P-R-N-A and place in one dish.
Inject another 20 to 30 embryos each with 20 picograms of Nain, RNA premixed with Y-F-P-R-N-A and place in dish. In addition, place 100 to 200 unin injected embryos per time point in another injection dish containing the FICO solution. Move these embryos in and outta the incubator at the same time as the injected embryos.
These are the hosts to be used for transplanting the caps after injection. Incubate all embryos at 14 degrees Celsius overnight. Repeat for all time points planned.
Following the overnight incubation, the animal cap can be isolated. Arrive early in the morning to stage the embryos. Remember to remove the unin injected host embryos from the incubator as well.
The earliest time point should be at stage nine. First, remove the embryos that did not survive. Proceed to isolate the animal caps from the viable embryos, working with each time point in batches to a cap isolation dish.
Add a generous amount of 0.7 XMMR plus gentamycin solution. Then pour off the fol solution from each plate containing the injected embryos and add back 0.7 XMMR plus Gentamycin solution. Dislodge the embryos from their injection dish wells using a transfer pipette and place in the freshly prepared cap isolation dish.
Use either the gastro master fitted with a yellow tip bent to 400 micrometers or a pair of sharp number five forceps to isolate animal caps in 0.7 XMMR plus Gentamycin solution. Keep two or three intact embryos for staging. After isolating the animal caps, put them back in the 14 degrees Celsius incubator along with the host embryos until the next day when the caps are transplanted onto the flank of the host embryos.
Remove caps and host embryos from the first time point from the 14 degrees Celsius incubator and stage the embryos sort for uniform fluorescent positive caps. Count the number of caps and remove. Double the number of host embryos to a freshly prepared injection dish with 0.7 XMMR plus Gentamycin solution with sharp number five forceps.
Remove the Lene membrane from each host embryo. Transfer them to the plate with the caps, but be careful without their lene membranes. Embryos can quickly dissociate at the water surface using the gastro master fitted with a yellow tip with a 200 to 250 micrometer bent wire at the highest setting or using number five forceps.
Remove a 200 micrometer square on the side of the embryo at stage 14 to 15. Then again using the gastro master or the number five forceps. Cut a cap in half and place the tissue in the hole made in the host embryo.
Rotate the embryo against the well to allow healing. Do this for as many embryos as you can while they're at stage 15. Repeat for all time points.
The embryo usually heals within 30 minutes. And with number five forceps, remove the dead cells which appear white with the gentle brushing motion. Grow the transplanted embryos at 18 degrees Celsius overnight.
After the overnight incubation, work under the dissecting fluorescence microscope to sort embryos with fluorescently labeled transplanted tissue. Put the selected embryos back at 18 degrees Celsius to grow until stage 42 to 43. When the animals reach the desired stage, anesthetize them in trica and examine under a fluorescence dissecting microscope and take pictures.
The transplanted embryos can then be fixed, sectioned and immuno stained, or one can perform in situ hybridization using markers for a tissue of interest. Here are representative results of a host tadpole with donor tissue expressing a fluorescent tracer and noggin one day after transplantation. Notice how the tissue balls up on the side of the tadpole.
The host embryos receiving controlled donor tissue expressing only the fluorescent tracer already start incorporating the animal cap cells onto their flank. A few days later, the tadpoles receiving nain expressing donor cells form eye like structures on their side while the tadpoles receiving the control donor tissue have fluorescent cells on the surface of their skin. We've just shown you how to perform the animal cap transplant assay in Zaps Lavis.
When doing this assay. It's important to remember to have everything ready prior to that day's procedure so the embryos don't develop faster than you can get to them. So that's it.
Thanks for watching and good luck with your experiments.
