A5177 5ea

Embryos were isolated from superovulated female mice (Myo10^flox/flox; Cre⁺ and Myo10^flox/flox; Cre⁻ as Myo10^−/− full mice, and Myo10^wt/wt; Cre⁺ and Myo10^wt/wt; Cre⁻ as control mice) mated with male mice (Myo10^wt/wt; Cre⁻). Superovulation of female mice was induced by intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (Ceva, Syncro-part), followed by intraperitoneal injection of 5 IU human chorionic gonadotropin (MSD Animal Health, Chorulon) 44–48 h later. Embryos were recovered at E0.5 from plugged females by opening the ampulla followed by a brief treatment with 37°C 0.3 mg/ml hyaluronidase (H4272-30MG; Sigma-Aldrich) and washing in 37°C FHM. When ampulla was not present, embryos were recovered by flushing oviducts with 37°C FHM (MR-122-D; Millipore) using a modified syringe (1400 LL 23; Acufirm). Embryos were handled using an aspirator tube (A5177-5EA; Sigma-Aldrich) equipped with a glass pipette pulled from glass micropipettes (Blaubrand intraMark or Warner Instruments). Embryos were placed in KSOM (MR-107-D; Millipore) supplemented with 0.1% BSA (A3311; Sigma-Aldrich) in 10 ml droplets covered in mineral oil (M8410; Sigma-Aldrich). Embryos were cultured in an incubator under a humidified atmosphere supplemented with 5% CO₂ at 37°C for 5 d. Embryos were scored for survival and embryonic stage from E0.5 to E4.5.

Zebrafish eggs were dechorionated 48 h post egg fertilization (48 hpf) with two forceps under a binocular microscope. Before cell injection, the dechorionated zebrafish were anesthetized with tricaine (0.042 mg/mL E3-medium) and then placed on a small sheet of wet paper in a Petri dish. Injection needles were prepared from glass capillaries (80 mm length, 1.55 mm diameter, from Hirschmann Laborgeräte GmbH, Eberstadt, Germany) with a needle puller to get needles with a tip of 0.02 mm in diameter. Needles were placed in a needle holder (Aspirator tube assemblies for calibrated microcapillary pipets from Sigma-Aldrich, A5177-5EA) with a rubber sleeve and a mouthpiece. Freshly prepared cell suspension (0.1–0.2 µL) was slowly injected into the left eye of the zebrafish using 70 to 90 cells (RB355 and WERI-RB-1 cells) or approximately 80 to 100 cells (Y79 cells). Successful injection was controlled by monitoring the green fluorescence of the cells. Cell injection into the left zebrafish eye was performed on twenty zebrafish for each of the cell lines. The un-injected right eye served as internal control and five completely un-injected zebrafish were used as negative control for each injection series. The experiments were approved by the German Ministry of Environment, Agriculture, Nature and Consumer Protection of North Rhine-Westphalia (LANUV) with the number 84-02.04.2016.A346.