All mice were maintained and used in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Japanese Association for Laboratory Animal Science and the National Research Institute for Child Health and Development (NRICHD) of Japan. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the NRICHD (Permit Number: A2006-009).
Adult female B6D2F1 mice were purchased from Clea Japan (Tokyo, Japan) and oocytes were collected following standard methods27 (link). PEs were generated using Ca-free M16 medium containing 8 mM SrCl2 and Cytochalasin B (5 μg ml−1) (Sigma-Aldrich, St Louis, MO, USA), and cultured KSOM (EMD Millipore, Darmstadt, Germany). Injection experiments (mRNA, short interfering RNA (siRNA) and nuclear transfer) were conducted using a Prime Tech Piezo drive (Sutter Instrument Company, Novato, CA, USA). To produce cloned embryos, nuclear-transferred oocytes were parthenogentically activated. Manipulated embryos were cultured to the developmental stages, as follows: 4-cell, 48 h; morula, 72 h; and blastocyst, 96 and 120 h after parthenogenetic activation or ICSI, respectively. All embryos were cultured at 37 °C in KSOM in an atmosphere containing 5% CO2. In the TSA experiment, the embryos were cultured for 24 h in activation and culture media containing 50 nM TSA (Sigma-Aldrich). IVF fertilization and nuclear transfer were performed following published procedures27 (link). To determine the effects of ectopic KDM4B expression on Xist expression in cloned embryos, doxycycline was added to ES cell culture and KSOM medium to a final concentration of 2 μg ml−1. Pseudopregnant ICR mice (Clea Japan) were used as embryo recipients. At E6.5, E9.5 and E18.5, the embryos were recovered from the uterus.
Adult female B6D2F1 mice were purchased from Clea Japan (Tokyo, Japan) and oocytes were collected following standard methods27 (link). PEs were generated using Ca-free M16 medium containing 8 mM SrCl2 and Cytochalasin B (5 μg ml−1) (Sigma-Aldrich, St Louis, MO, USA), and cultured KSOM (EMD Millipore, Darmstadt, Germany). Injection experiments (mRNA, short interfering RNA (siRNA) and nuclear transfer) were conducted using a Prime Tech Piezo drive (Sutter Instrument Company, Novato, CA, USA). To produce cloned embryos, nuclear-transferred oocytes were parthenogentically activated. Manipulated embryos were cultured to the developmental stages, as follows: 4-cell, 48 h; morula, 72 h; and blastocyst, 96 and 120 h after parthenogenetic activation or ICSI, respectively. All embryos were cultured at 37 °C in KSOM in an atmosphere containing 5% CO2. In the TSA experiment, the embryos were cultured for 24 h in activation and culture media containing 50 nM TSA (Sigma-Aldrich). IVF fertilization and nuclear transfer were performed following published procedures27 (link). To determine the effects of ectopic KDM4B expression on Xist expression in cloned embryos, doxycycline was added to ES cell culture and KSOM medium to a final concentration of 2 μg ml−1. Pseudopregnant ICR mice (Clea Japan) were used as embryo recipients. At E6.5, E9.5 and E18.5, the embryos were recovered from the uterus.
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