Ecfg21

Mature COCs from JNPs ovaries were pipetted to DPBS solution containing 0.1% hyaluronidase (H3506). MII oocytes were discovered in sucrose media after being washed three times in T-HEPES medium. They were removed from the enucleation media (T- HEPES with cytochalasin, Sigma-Aldrich C6762 7.5 g/mL) with a microinjection pipette to remove the polar body and MII chromosome. The donor cell was inserted after approximately an hour of rest. After being injected with donor cells, the oocytes were transferred to a 1 mm fusion chamber containing a 0.28 M mannitol solution. A cell manipulator (ECFG21; Nepa Gene, Chiba, Japan) was used to apply 130 V and 50 ms⁻² pulse to activate electrolysis. The reconstructed oocytes were cultured in porcine zygote medium-5 at 39 °C and 5% CO₂ after fusion and activation. The fusion was examined the next day. The reconstructed embryos were implanted into recipient JNPs. Approximately 170 reconstructed embryos were surgically transferred after being cultivated for 1 to 2 days. The fallopian tube was implanted with embryos, the wound was sutured, and, 30 days following the transplant, an ultrasound machine was used to check for pregnancy. Regular checks were conducted throughout the pregnancy, and the offspring was delivered naturally.

Eight-week-old female BALB/c mice were immunized every 2 weeks for a total of six times and then boosted twice in a week. Fifty micrograms of antigen was prepared with an equal volume of TiterMax Gold adjuvant (Sigma-Aldrich) according to the manufacturers’ instructions. Four days after boosting, the splenocytes of immunized mice were collected and fused with SP2/O myeloma using electro cell fusion generator ECFG21 (Nepa Gene) according to the manufacturers’ instructions. The fused cells were cultured in GIT/IL-6/HT supplements and aminopterin medium [GIT medium (FUJIFILM Wako) supplemented with recombinant human interleukin-6 (IL-6) (1 ng/ml; PeproTech), hypoxanthine thymidine (HT) supplement (Gibco), and 0.4 μM aminopterin (Sigma-Aldrich)] for 1 week to select hybridomas. We performed enzyme-linked immunosorbent assay, WB, and IP to screen hybridomas using culture supernatant. Serial dilution was performed to monoclonize selected hybridomas. Monoclonal hybridomas were cultured in GIT medium (FUJIFILM Wako) supplemented with IL-6 (1 ng/ml) for antibody production. The isotype of antibodies was determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche). The animal experiments were approved by the Animal Care and Use Committee of Keio University and were conducted in compliance with the Keio University Code of Research Ethics.

After confirming induction of serum antibodies against AMIGO2-Ig protein, spleens and lymph nodes from immunized rats were harvested from euthanized rats, homogenized to single-cell suspensions, and fused with myeloma P3X63Ag8.653 cells using an electro-cell-fusion generator (ECFG21; Nepagene, Chiba, Japan). Fused hybridoma cells were plated in 96-well plates. After approximately 14 days of culture, a primary screen of supernatants was performed by ELISA. Hybridoma clones producing AMIGO2-specific Abs were identified by ELISA using GST-AMIGO2-Ig following HAT selection. Positive wells were picked and passaged in 96-well plates. Each supernatant was reanalyzed by ELISA using Tag, Trx-AMIGO2-Ig, and GST-AMIGO2-Ig. Hybridoma clones that reacted with Trx-AMIGO2-Ig and GST-AMIGO2-Ig, but not Tag, were established using two or more limited dilutions.