Making Gynogenetic Diploid Zebrafish by Early Pressure

Hi, I am Charlene Walker from the lab of Charles Kimmel at the University of Oregon, the Institute of Neuroscience. And I'm Gregory Walsh. I work in the lab of Cecilia Moens in the Division of Basic Sciences at the Fred Hutchinson Cancer Research Center.

Today we will show you a procedure for making tic diploid zebrafish by the early pressure method. This method uses hydrostatic pressure to depolymerize microtubules in an egg fertilized with UV irradiated sperm. Done with the right timing.

This prevents the expulsion of the second polar body generating a diplo embryo whose homologous chromosomes are the two products of meiosis.One. This technique was worked out in the early 1970s when I worked in the lab of George Reisinger at the University of Oregon. In our lab, we use this procedure in two generation four genetic screens.

The gametes of F1 mutagen fish can be screened directly for recessive mutations that cause developmental phenotypes in homozygous gyno, genetic diploid offspring. Since zebrafish exhibit high genetic interference, we also use this method to map mutations to chromosomes and to estimate how far these mutations lie from the centromere. So let's get started During the late afternoon, the day before the experiment, prepare for sperm collection by separating males and females to be squeezed, old males can be used since they will not be contributing any genetic material.

Females should look large and fat in the belly while males should look yellow and spry. Hold the males and females overnight in separate holding tanks where they can be easily accessed in the morning. On the morning of the experiment, prepare Hank's solution and add sodium bicarbonate.

Bring to the fish facility in an ice bucket. The Hank's solution trica a timer and tubes for collecting sperm. Finally, once in the fish facility, dilute the trica in fish water.

This will be used to anesthetize the fish in the next step. To begin sperm collection, prepare a clear vial of Hank's solution on ice. Chilly enough hanks for about 50 microliters per male to be squeezed anesthetize one or two males at a time.

By immersion and trica, they can be lifted out of the anesthetic with a plastic spoon. Rinse the males and fish water and blot damp dry on a paper towel. This is very critical because water activates sperm.

Once they are dry, mount the fish in a foam holder under a stereo microscope at low magnification with epi illumination. Separate the pelvic fins with forceps and position a micro capillary at the opening of the cloaca to begin collecting sperm. Stroke the sides of the fish gently but firmly with smooth mill Pour forceps as the milky sperm comes out of the genital pore collected in the micro capillary using gentle suction, pull the sperm from several males in ice.

Cold hank saline Sperm from five to 10 males is adequate for fertilizing eggs from 20 to 30 females. Sperm kept in cold. Hanks will continue to fertilize eggs efficiently for several hours.

Collect enough sperm to make a cloudy suspension to UV inactivate sperm. Start by filling the bottom of a glass Petri dish with ice. Put a watch glass on top of the ice in the dish.

Next, use the pipette to transfer the sperm from the test tube onto the watch glass. Be sure to get as much of the sperm into the pipette as possible without taking up any bubbles of air. It is critical to avoid bubbling the sperm, expel the sperm in a thin even layer on the watch glass and discard the pipette.

After the sperm has been transferred, place the Petri dish into a UV crosslinker. Turn on the Crosslinker set on energy to deliver 900 JUULs. After cross-linking, use a clean pipette to remove the sperm from the watch glass and transfer it to an fendor tube marked UV sperm and place on ice.

To begin egg collection first anesthetize two females at a time. In Trica, it is critical not to keep the fish in trica too long because prolonged anesthesia can kill the fish. Next, rinse the fish in fish water and blot damp dry on a paper towel.

This is critical because excess water will swell the eggs and prevent fertilization. Place the female in a 35 millimeter plastic Petri dish with damp but not wet fingers. Press gently but firmly on the belly.

If she is ready to lay eggs, they will come out easily gather the eggs with a spatula and return the female to water. Good eggs are a yellowish, translucent color, whereas eggs that have remained in the female too long are white and watery. To ensure getting good eggs, collect them during the first two hours after the lights come on in the fish facility, keep the eggs covered to prevent drying out.

After collecting the eggs, add 30 to 50 microliters of the UV sperm suspension to the eggs and stir gently with a pipette tip. Immediately add about one milliliter of fish, water and start the timer. Eggs left to develop at this point will be haplos.

The following steps must be accomplished within 90 seconds of fertilization. Start the timer. At the moment that eggs are activated by the addition of water.

The exact timing of the metaphase of meiosis one is temperature dependent. The timing of pressure on and off is optimized for 28.5 degrees Celsius. After starting the timer, wait a few moments, then add more water and sw the eggs to the center of the dish.

Using a cutoff and polished pasture pipette. Transfer the fertilized eggs to the pressure vials if needed, add more egg water to the vial so they're almost overflowing, leaving a bead of water at the loop of the vials. Next place a rubber top over the bead of water and secure the plastic lid over the rubber.

Place the vials upside down in the pressure cylinder, adding more egg water. To refill the cylinder, put the top on the cylinder securely. Once closed, place the cylinder in the press with the plunger up and apply pressure of 8, 000 pounds per square inch.

Leave the cylinder in the press until six minutes after the time of activation for a total time of 4.5 minutes of pressure. Six minutes after activation, remove the cylinder from the press. Remove the vials from the cylinder and carefully dry them to prevent cooling of the embryos.

Reset the cylinder and refill it with egg water. Prepare the next vials by partially filling them with egg water as needed. Repeat these steps for all the batches of eggs.

The cylinder will hold up to two vials at a time, so you may want to delay fertilization of eggs one to two minutes to see if other females already in the trica give eggs. This will help speed things up by treating more batches at one time. Also, if you squeeze fish while the press is being used, the eggs may be too dry to be viable by the time the press can be used again, 24 hours after fertilization, a haploid zebrafish embryo will have a short, stocky body, a short tail, and less clearly formed brain.

Yet they will all have the same body parts such as somites, no cord, eyes and ears. As diploids, haplos, and diploids all exhibit uninhibited flexing motions around 24 hours. Post fertilization, EP diploid siblings have long tails, nicely sculptured brains, and should be about 60 to 70%viable.

These look very much like the untreated deployed controls. Usually a batch of haplo embryos will have a mixture of good looking embryos, dead embryos, and everything in between. The proportion of good looking embryos to defective ones reflects the deleterious genes harbored in the female genome.

Mutations are relatively easy to identify even with this background because there will be several individuals up to 50%with the same characteristic phenotype. It is important to make sure that the embryos produced by this method are true tic OIDs as controls generate a clutch of normal deployed eggs by keeping aside some of the sperm without UV inactivation and a clutch of haplo embryos by keeping aside a clutch of eggs fertilized with UV sperm without going through the EP procedure, haplos are not viable after two to three days. EP deployed should look like normal diploids.

Although some irregular EP monsters may occur, clutches of EP diploids should be largely viable around 70 to 90%The presence of haplos in the EP deployed clutch suggests a failure in the EP process. For example, the press failing to hold pressure. The presence of diploids in the haplo clutch suggests incomplete sperm inactivation, for example, a failure in the crosslinker.

We've just shown you how easy it is to do in vitro fertilization with zebrafish and how to make gyno genetically derived diploid zebrafish embryos by inhibiting the second myonic division. When doing this procedure, it's important to remember that the timing of the early pressure is precise and that the press holds its pressure at 8, 000 pounds per square inch throughout the treatment. Be sure to have your press calibrated.

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