Human chorionic gonadotropin (hcg)

WKY/NCrl rats were obtained from Charles River, and DA/Slc rats were purchased from SLC Japan. The rats were kept with a 12:12-h light: dark cycle.
To investigate the electroporation efficiency (Fig. 1), rat females (WKY and DA) were super-ovulated by intraperitoneal injection of 20 IU pregnant mare serum gonadotropin (PMSG; ASKA Pharmaceutical Co. Ltd., Tokyo, Japan) and 10 IU human chorionic gonadotropin (hCG; ASKA Pharmaceutical Co. Ltd.). The super-ovulated females were mated to rat males (WKY and DA) overnight. To analyze CRISPR/Cas9 systems (see Figs. 2 and 3), estrous female rats were mated to male rats without super-ovulation. Presence of copulation plugs was confirmed by visual inspection in the following morning and used for the electroporation experiments.
All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal works were approved by the appropriate committee.

Nuclear transfer was carried out as described0 . Briefly, BDF1 female mice were induced to superovulate by the injection of 7.5 IU of pregnant mare serum gonadotropin (Sankyo Yell Yakuhin, Co., Tokyo, Japan) and 7.5 IU of human chorionic gonadotropin (hCG; ASKA Pharmaceutical Co., Ltd, Tokyo, Japan) at a 48 h interval. At 15 h after the hCG injection, cumulus–oocyte complexes were collected from the oviducts and the cumulus cells were dispersed in potassium-enriched simplex optimized medium (KSOM)8 (link) containing 0.1% bovine testicular hyaluronidase. Collected cumulus cells were used as nuclear donors. Immature Sertoli cells were collected as described previously and used as nuclear donors9 (link). The oocytes were enucleated in Hepes-buffered KSOM containing 7.5 μg/ml cytochalasin B. The donor nuclei were injected into enucleated oocytes using a Piezo-driven micromanipulator (PMM-150FU, Primetech, Ibaraki, Japan). After culture in KSOM for 1 h, the injected oocytes were activated in Ca²⁺-free KSOM containing 2.5 mM SrCl₂ for 1 h. The reconstructed embryos were cultured in KSOM containing 5 μg/ml cytochalasin B for 5 h. For TSA treatment, 5 nM was added into each medium from activation for 8 h.

ICR (CD1) female mice were used as oocyte donors and superovulated via treatment with 7.5 International Unit (IU) of equine chorionic gonadotropin (ASKA Pharmaceutical, Tokyo, Japan) and 7.5 IU of hCG (ASKA Pharmaceutical) administered 48 h apart. At 16 h after hCG administration, cumulus oocyte complexes were collected from oviducts and treated in M2 medium19 containing 0.13% hyaluronidase (Sigma‐Aldrich, St. Louis, MO) to remove cumulus cells. After washing in hyaluronidase‐free M2 medium, the denuded MII oocytes were used for in vitro fertilization (IVF) immediately after retrieval from oviducts (0 h‐oocytes). IVF was performed by modifying the methods described in previous studies.19, 20 For IVF using postovulatory‐aged oocytes, the 0 h‐oocytes were further incubated in M16 medium21 in a humidified atmosphere of 5% CO₂ at 37°C for 12 or 24 h (12 h‐ and 24 h‐oocytes, respectively).