6 dmap

PLGA (Sigma-805726), TRITC (Sigma-87918), RES (Sigma-R5010), PVA (Sigma-P8136), Dichloromethane (Sigma-650463), PBS (Sigma-2272), TCM-199 (Gibco-C11150500BT), BME AA (Sigma-B6766), MEM AA (Sigma-M7145), Glutamine (Sigma-G8540), BSA (Sigma-A1933), DMSO (Sigma-D2650), FSH (Solarbio-F8470), LH (Solarbio-L8040), FBS (Gibco-16000), Gentamicin (Merck-G1264), NaHCO₃ (Wako-191-01305), HEPES (Sigma-H4034), Sodium Pyruvate (Wako-199-03062), E₂ (Sigma-E2758), DPBS (Sigma-D8662), Ethylene Glycol (EG) (Sigma-324558), Trehalose (Sigma-T0167), 6-DMAP (Sigma-D2629), H₂O (Sigma-W1503).

To obtain female embryos solely for the sex ratio determination, parthenogenetically activated embryos were generated. Matured bovine oocytes were stripped from cumulus cells and placed in fertilization medium as described above. The fertilization medium was further supplemented with 5 mM Ionomycin (Sigma-Aldrich) and oocytes were incubated for 5 min. After three times washing in SOF the oocytes were transferred to SOF supplemented with 1.9 mM 6-DMAP (Sigma-Aldrich) and incubated for 3.5 hrs in a humidified atmosphere with 5% CO₂ and 20% O₂ at 39°C. Hereafter, the oocytes were washed three times in SOF and cultured in SOF in a humidified atmosphere with 5% CO₂ and 7% O₂ at 39°C. At day 5 of culture, cleaved embryos were transferred to fresh SOF and cultured for an additional 4 days when blastocysts were collected for DNA extraction.

The reconstructed oocytes were parthenogenetically activated by incubation in 7.5 mol/L ionomycin (I3909, Sigma, St Louis, MO, USA) for 10 min followed by incubation in 2 mmol/L 6-dimethylamino purine (6-DMAP; d2629, Sigma, St Louis, MO, USA) for 4 h. Activated oocytes with 1 PN were injected G1 gRNA, Cas9 mRNA, and the ssDNAoligo into the cytoplasm 5–6 h after activation. The survived reconstructed embryos were cultured in microdrops of G-1 medium (Vitrolife, VitrolifeSweeden AB Göteborg, Sweeden) at 37°C in a humidified atmosphere of 6% CO₂, 5% O₂, and 89% N₂ for 42 h. Blastomere of reconstructed embryos were individually aspirated out of ZP by the pipette.

Electrical fusion of oocytes after nuclear transplantation was performed by applying the experimental method of Ryu and Yoon (2018) and energizing 1.1 kv/cm of direct current for 30 μsec in 280 Mm mannitol containing 0.1 mM CaCl₂, 0.1 mM MgCl₂, with calcium-free TLH by inducing cell fusion. Fused nuclear embryos were incubated for 4 h in a culture medium supplemented with 2 mM 6-dimethylaminopurine (6-DMAP; Sigma) and 7.5 μg/ml cytochalasin B to induce activation (Fig 1).

Oocytes were activated using calcium ionophore A23187 (25 μM, 5 min; Sigma-Aldrich, USA) combined with a 6-dimethyl aminopurine - 6-DMAP (2 mM, 2 h; Sigma-Aldrich, USA) treatment [27 (link)]. In experimental groups, porcine oocytes were exposed to prolonged cultivation (aging) for 24 hours in medium M199 supplemented with hydrogen sulfide donor, Na₂S.9H₂O in concentrations 150 μM, or 300 μM. These concentrations were selected after previous testing, because in the group of porcine oocytes aged 72 hours in medium supplemented with 150 μM, or 300 μM of Na₂S.9H₂O, fragmented oocytes failed to occur (see Supporting Information, S1 Table).
Parthenogenetically activated oocytes were subsequently cultivated 24 hours. Oocytes with formed pronuclei were considered parthenogenetically activated. Experiments were repeated three times and a minimum of 120 oocytes were evaluated in each experiment.

The matured oocytes were first treated with 5 μM ionomycin in M2 medium for 7 min, and then cultured for 4 h in CZB medium (EasyCheck, M1650) supplemented with 2 mM 6-DMAP (Sigma, D2629) and 5 mg/l cytochalasin B (MedChemExpress, HY-16928), at 37 °C in a humidified atmosphere containing 5% CO₂ in air. Following activation, the oocytes were washed and transferred to CZB droplets for subsequent culture. After 24 h, oocytes were observed under an inverted microscope for activation assessment. Oocytes showing two symmetrical cells each having a nucleolus were judged as activated. Activated embryos were further cultured in CZB and the cleavage rate and blastocyst formation rate were assessed at 48 h and 108 h, respectively.

The female ICR mice (6–8 weeks old) were acquired from the School of Medical Science, Jilin University. Superovulation was induced in female mice by an intraperitoneal administration of 10 IU pregnant mare’s serum gonadotropin (PMSG; Merck Millipore) and was injected with 10 IU human chorionic gonadotropin (hCG; Sigma-Aldrich, St. Louis, MO, USA)) 48 h later. PA embryos production was performed according to the procedure described by us previously. Briefly, the oviducts of female mice were carefully removed, and cumulus-oocyte complexes (COCs) were extracted from the oviducts as unfertilized oocytes after post-PMSG and -hCG injection. Oocytes were then denuded from their cumulus cells by briefly exposing the oocytes at the metaphase stage of the second meiotic division (MII) to a serum-free medium comprising hyaluronidase (Sigma). The oocytes were then treated with a calcium ionophore (ionomycin calcium salt; Sigma) for 5 min. Parthenogenetic activation was achieved by incubating the unfertilized oocytes in M16 medium supplemented with 6-DMAP (2 mmol/l; Sigma) for 4 h. Subsequently, these unfertilized oocytes were transferred to the fresh M16 medium. PA activation was established by the presence of two pronuclei, which developed to the two-celled stage. The embryo culture was conducted until the blastocyst stage.