Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains

Drosophila strain must be maintained by the frequent transfer of adult flies to new vials. This creates a danger of mutational deterioration and genetic changes. We aim to develop an alternative method for the long-term preservation of Drosophila strains without such changes.

Compare with in vivo editing, genome editing of primordial germ cells, or PGCs, could increase knock-in inefficiency and make more complicated genomic changes. Thus, a wide application of PGC manipulation facilitates genetic engineering in many fields. We now focus on xenotransplantation.

By using Drosophila melanogaster as a surrogate host, we have succeeded in reviving stock of cell species in the melanogaster species subgroup. By addressing xenotransplantation barriers and finding possible solutions, we would like to explore the potential application of PGC transplantation and cryopreservation. Proceed to collect the primordial germ cells, or PGCs, from the Drosophila embryos after assembling the micromanipulator system.

While working under a stereo microscope, use forceps to transfer the embryos. Align the dechorionated embryos in two rows on a PGC-collection glass slide along two reference lines. Orient the embryos with their anterior to the right and ventral side up.

Gently move the transplantation needle tip to the anterior end of an embryo. Penetrate the embryo by moving the microscope stage and retract the needle slightly when the tip reaches the posterior end. Discharge any yolk in the needle inside the somatic cell layer.

Move the needle tip to the posterior pole and gently load the PGCs into the needle without taking much time. Pull the needle out of the embryo quickly. Discharge yolk and other contaminants from the needle while keeping the PGCs.

Load clean silicone oil from the pool into the needle. Before collecting PGCs from a new embryo, deposit as much silicone oil as possible inside the somatic cell layer while keeping the loaded PGCs in the needle. After collecting PGCs from all embryos in a row, deposit the PGCs onto the surface of an embryo and unload any yolk or other contaminants onto another neighboring embryo.

In a similar manner, collect and combine PGCs from the embryos in the second row. Proceed for the cryopreservation of the primordial germ cells or PGCs. After collecting and depositing the PGCs from the donor embryos on the surface of an embryo.

Wash the transplantation needle with a cryoprotective agent, or CPA, and load fresh CPA into the needle. Add the loaded CPA to the PGCs deposited on the embryo, while ensuring the volume of CPA is equivalent to that of the PGCs. Remove excess CPA from the cluster of PGCs one to two seconds after CPA addition.

Next, empty the needle and load it with silicone oil for five seconds or longer. Load all the collected PGCs into the needle, followed by loading silicone oil again for five seconds or longer. Ensure the PGCs are sandwiched between two layers of silicone oil.

Detach the needle from the micromanipulator. Using soft tissue paper, blot the oil off the needle surface without touching the needle tip with the tissue. Attach the needle to a needle holder and using vinyl tape, lock it in position at the base.

Flash freeze the holder with the needle pointing downwards by submerging it in liquid nitrogen. Do not release the holder until the liquid stops fizzing out of the rack. Store the holder in a liquid nitrogen storage tank in the liquid phase area.

To begin, align the stage five agametic Drosophila host embryos on a transplantation glass slide. While orienting their posterior to the right and ventral to the top, line up approximately 30 embryos in two rows in 20 minutes. Thaw the cryopreserved PGCs in the needle by submerging the holder with the needle pointing downward into EBR solution at room temperature.

Keep it submerged for 10 seconds. Place the transplantation glass slide on the microscope stage. Attach the freeze thawed needle to the capillary holder and bring the first embryo into the left row and the needle tip into the same focal plane.

Using a 20X objective lens, position the needle tip gently at the surface of the posterior end of the embryo. Prod the outside of each embryo gently and confirm that they slowly return to their original shape. Penetrate an embryo from the posterior pole using the needle.

Gently deposit approximately 10 to 20 PGCs inside the posterior pole, specifically between the vitelline membrane and the somatic cell layer and retract the needle from the embryo before repeating the same for subsequent embryos. Finally, to revive the strain, cross newly emerged females and males.