Mouse zygotes were collected from superovulated BCF1 (C57BL/6 × CBA/CA) females as described previously (28 ). Briefly, female BCF1 mice at 5 weeks of age were injected with 5 IU of pregnant mare serum gonadotrophin (PMSF), followed by 5 IU of human chorionic gonadotropins (hCG) 48 h apart, and mated with male mice. Successful mating was determined the following morning by detection of a vaginal plug. Mouse zygotes were transferred to M2 medium (Sigma) containing 0.1% (w/v) hyaluronidase to remove cumulus cells and cultured in M16 medium (Sigma) at 37°C, 5% CO2 in air (29 (link)).
Microinjection was performed 24 h after human chorionic gonadotropin injection (30 (link)). To visualize the pronuclei, cumulus-free zygotes were centrifuged at 13,000 rpm for 5 min. Linearized GFP-Dnmt1sc− or GFP-Dnmt1oc− expression construct (4–20 ng/μl) was microinjected into the male pronuclei of mouse zygotes using an inverted microscope equipped with a micromanipulator (Leica). The injected zygotes were then cultured in M16 media for 48 h. The resulting four- or eight-cell stage embryos were collected, fixed using 4% formaldehyde and mounted with DAPI (Vector). The localization of Dnmt1 isoforms was evaluated by detecting GFP signals under a fluorescence microscope equipped with a microtome (Zeiss).
Microinjection was performed 24 h after human chorionic gonadotropin injection (30 (link)). To visualize the pronuclei, cumulus-free zygotes were centrifuged at 13,000 rpm for 5 min. Linearized GFP-Dnmt1sc− or GFP-Dnmt1oc− expression construct (4–20 ng/μl) was microinjected into the male pronuclei of mouse zygotes using an inverted microscope equipped with a micromanipulator (Leica). The injected zygotes were then cultured in M16 media for 48 h. The resulting four- or eight-cell stage embryos were collected, fixed using 4% formaldehyde and mounted with DAPI (Vector). The localization of Dnmt1 isoforms was evaluated by detecting GFP signals under a fluorescence microscope equipped with a microtome (Zeiss).