Mouse embryos were collected from 4-6 week old superovulated CB6F1 female mice (BALB/c × C57BL/6; Charles River, Wilmington, MA). For RNAi, zygotes at the pronuclear stage (i.e. prior to fusion of the paternal and maternal genetic material in the first cell cycle) were injected with 400-700bp long double-stranded RNA corresponding to target gene sequences. Control (untreated or mock-injected) and RNAi-treated zygotes were cultured in a constant environment of 37°C, 5% CO2, and 95% humidity for up to 120 hours (5 days), during which time images were captured every 420s (at 7 minute intervals). Under these conditions, control embryos consistently achieved > 95% development to the blastocyst stage.
Time-lapse recordings were collected using an Eclipse TI inverted microscope (Nikon Instruments, Inc., NY) with Hoffman modulation optics, a heated stage-top incubator (Tokai Hit Co., Ltd., Japan) with an independent air circulation and heating Air-Therm ATX system (WPI, Inc., FL) mounted on an XYZ motorized stage (Nikon), and NIS Elements imaging software (Nikon) running on a Dell Precision Workstation T7400.
Time-lapse recordings were collected using an Eclipse TI inverted microscope (Nikon Instruments, Inc., NY) with Hoffman modulation optics, a heated stage-top incubator (Tokai Hit Co., Ltd., Japan) with an independent air circulation and heating Air-Therm ATX system (WPI, Inc., FL) mounted on an XYZ motorized stage (Nikon), and NIS Elements imaging software (Nikon) running on a Dell Precision Workstation T7400.