The overall goal of this procedure is to promote diploidization in the early zebrafish embryo through inhibition of cytokinesis corresponding to one embryonic cell cycle. This method can help answer key questions in the field of genetics such as through the identification of genes involved in adult or reproductive traits, the generation of inbred lines or other genetic manipulations. The main advantage of this technique is that it is simple yet effective in promoting diploidization, allowing the direct homozygosis of alleles present in a single heterozygous individual.
The afternoon before the experiment, set up mating pairs of the desired zebrafish strain in standard zebrafish mating boxes with dividers to separate males and females. The morning of the experiment, remove the physical partition for egg laying to begin. Visually inspect tanks containing mating pairs to detect extrusion of eggs during natural mating.
At the first signs of egg extrusions, separate the male and female to interrupt breeding. Keep the pre-selected females either separately or pooled in the same tank. Use multiple females depending on the number of embryos desired.
To prepare a sperm solution from zebrafish testes, after confirming euthanasia according to the text protocol, remove the males from the beaker. Use conditioned water to rinse briefly and dry them lightly by briefly placing them on several locations of paper towel. Use dissecting scissors or a razor blade to decapitate the fish and make a longitudinal cut along the abdomen.
Then under a dissecting microscope with a reflected light source, use dissecting forceps to remove the internal organs. Pull out each of the testes'masses and transfer them to a microcentrifuge tube with Hank solution. To release the sperm into the solution, use a narrow spatula to shear the testes or use a 1, 000 microliter tip and pipette up and down five to six times in the solution while avoiding air bubble formation.
Then allow the testes debris to settle. Store the sperm solution on ice where it can keep its potency for two to three hours. If proceeding to UV sperm treatment, transfer the sperm solution to a clean microcentrifuge tube leaving the sperm debris behind.
To carry out UV treatment of sperm, transfer up to one milliliter of the sperm solution to a clean dry well of a depression glass plate sitting on an ice bed. Place the ice bed with the depression plate under a UV lamp at a distance of 19 centimeters from the light source and treat the sperm solution at 115 volts for 90 seconds. With the end of an unused pipette tip, gently mix the solution every 30 seconds during UV treatment.
Using a clean pipette tip on a micropipette, transfer the treated sperm solution to a new microcentrifuge tube. Store on ice no longer than two to three hours until needed for IVF. After anesthetizing female zebrafish according to the text protocol, as soon as the fish stops gill movement, use a spoon to collect the fish and use conditioned water to briefly rinse it.
With the spoon moving from the anterior to the posterior of the fish to avoid damaging the gill operculum, place the fish briefly and repeatedly on several locations of a paper towel to lightly dry it. Then transfer it to a clean 10 centimeter diameter Petri dish. Use lab wipes or soft tissue to further gently dry the anal fin area to prevent any released eggs from prematurely being activated by water.
Then after lightly dampening the fingers with water, place one finger of one hand on the female's back as support and with the finger on the other hand, apply slight pressure along the female's abdomen until eggs are extruded. It is crucial that the extruded eggs do not encounter any excess water to avoid them from becoming prematurely activated which would prevent them from becoming fertilized by sperm. Use a narrow spatula to move the eggs away from the female's body.
Then place the female back into a tank with conditioned water for recovery. Within 90 seconds after egg extrusion, add 100 microliters of sperm solution to the extruded eggs in a Petri dish. Gently swirl the pipette tip among the eggs to mix the sperm and eggs together, being sure not to lift the tip from the surface of the Petri dish to avoid egg damage.
Immediately activate the eggs by adding approximately one milliliter of embryonic or E3 medium and again gently mix the eggs and sperm by using a pipette tip to swirl. Initiate timing relative to fertilization. At one minute post fertilization in the one milliliter volume, use E3 to flood the plate.
Leave the fertilized eggs undisturbed until 10 to 12 minutes post fertilization or MPF to allow egg activation including full chorion expansion. After expansion of the chorions, pour the embryos from the Petri dish into a tea strainer. Then use a wash bottle with E3 to rinse the Petri dish to collect any remaining eggs into the strainer.
Place the tea strainer with the embryos inside a beaker in a pre-heated bath with E3 at 28.5 degrees Celsius. Pre-equilibrate the beaker and E3 to the water bath temperature and make sure there is enough E3 so that all embryos in the strainer are submerged. At 22 MPF, remove the tea strainer from the water bath then briefly blot it onto a paper towel to remove excess moisture and place it inside a similarly pre-heated E3 beaker in a heat bath at 41.4 degrees Celsius.
The key step in the procedure is the heat shock which induces a one-cycle delay in the cell division and therefore hold genome duplication. At 24 MPF, briefly blot the strainer and transfer it back into the E3 in a 28.5 degrees Celsius water bath. Then at 29 MPF, use a wash bottle with E3 to transfer the embryos from the tea strainer to a 10 centimeter Petri plate.
Between 35 to 45 MPF, under a dissecting microscope with a transmitted light source, select embryos that are undergoing symmetrical cleavage into the two-cell stage and which were therefore fertilized. Remove embryos that are not undergoing cell cleavage. Continue observing the fertilized embryos selected for normal cell division during the first cell cycle and during the second cell cycle.
Sort embryos according to the following categories. Four cells or no stall in the two to two arrangement standard for a four-cell embryo, three cells or partial stall in an aberrant two smaller cells to one large cell arrangement and two cells or stalled embryos. Sort the stalled embryos into a fresh Petri plate.
Allow up to 80 embryos to develop in the Petri dish. At 24 hours post fertilization or HPF, observe the embryos to determine whether they have a normal morphology characteristic of diploid or homozygous diploid embryos. In contrast to reduced axis extension, an increased body thickness characteristic of haploids.
In addition, at 36 HPF, assess diploidization using genetic pigment markers and other assays such as golden or albino. Finally, remove any embryos that appear to have a haploid morphology or that have lysed or exhibit other grossly abnormal defects. The derived method demonstrated here, referred to as HS2, uses a heat shock during the period 22 to 24 MPF and induces cytokinesis arrest during the second embryonic cell division at 50 MPF.
During the stall, ongoing DNA synthesis result in embryos undergoing diploidization from either haploid to diploid or diploid to tetraploid. HS2 treated embryos can exhibit a variety of blastomere arrangements including no stall where neither blastomere exhibits a delay, a partial stall where one of the two blastomeres is delayed and stalled where both blastomeres exhibit delays. The morphology of the embryos can be determined at 24 HPF with a normal morphology characteristic of diploid or homozygous diploid embryos or the shorter and wider body axis morphology characteristic of haploid embryos.
If properly selected for a one-cell cycle delay, all embryos should exhibit a normal diploid morphology. Once mastered, this technique can be done in two hours if it is performed properly. Although, it will take more time to process multiple clutches.
While attempting this procedure, it is important to remember to separate females in time, dissect testes accurately, avoid prematurely contact of extruded eggs with water and keep proper timing of the fertilized clutches. Following this procedure, other methods like forward genetic screens can be performed in order to answer additional questions like what genes are involved in specific biological functions. After its development, this technique paved the way for researchers in the field of genetics to explore traits relevant to juvenile, adult and intergenerational traits in the zebrafish.
After watching this video, you should have a good understanding of how to use heat treatment to induce diploidization in zebrafish. Don't forget that working with UV light can be extremely hazardous and precautions such as wearing UV protective safety glasses should always be taken while performing this procedure.
