Microinjection of Oocytes with Regulators

Oocytes still enclosed in the cumulus cells (CEOs) were injected with 12.5 ng/μl Il7-RL, Thumpd1-RL, or Tpx2-RL and 12.5 ng/µl of polyadenylated FL using a FemtoJet Express programmable microinjector with an Automated Upright Microscope System (Leica, DM4000B). Injected CEOs were pre-incubated for 3 h in culture medium supplemented with 2 μM milrinone, and then cultured in milrinone-free medium supplemented with 100 nM amphiregulin. After 16 h, the CEOs were denuded, collected in lysis buffer and frozen. Luciferase activities in the oocyte extracts were measured using a dual-luciferase reporter assay kit (Promega), and luminescence was detected with a SpectraMax L luminometer (Molecular Devices). Data are reported as ratios of RL and FL.