Ncaph2tm1a(EUCOMM)Wtsi (MBCH;EPD0070-2-G090) were obtained from the Wellcome Trust Sanger Institute. The corresponding flox allele was obtained as previously described (Houlard et al., 2015 (link)). The fusion was obtained by cloning in frame Smc2 cDNA, a linker containing three TEV protease cleavage sites (GGGGSGGGSGGGGTGSENLYFQGPRENLYFQGGSENLYFQGTRGGGGSGGGGSGGGG ), Ncaph2 cDNA (Origene, MC200537) and the eGFP ORF in the pUC19 vector.
Fully grown prophase-arrested GV oocytes were isolated and injected with mRNAs (5–10 pl) diluted in RNase-free water at the following concentrations: H2b–mCherry: 150 ng μl−1, Mad2: 200 ng μl−1, NCAPH2–Gfp: 50 ng μl−1, Fusion-EGFP (50 ng μl−1), Mcph1 (200 ng μl−1), Tev protease: 250 ng μl−1. All experimental procedures were approved by the University of Oxford ethical review committee and licensed by the Home Office under the Animal (Scientific procedures) Act 1986. No statistical method was used to predetermine sample size. The experiments were not randomised and the investigators were not blinded to allocation during experiments and outcome assessment.
Fully grown prophase-arrested GV oocytes were isolated and injected with mRNAs (5–10 pl) diluted in RNase-free water at the following concentrations: H2b–mCherry: 150 ng μl−1, Mad2: 200 ng μl−1, NCAPH2–Gfp: 50 ng μl−1, Fusion-EGFP (50 ng μl−1), Mcph1 (200 ng μl−1), Tev protease: 250 ng μl−1. All experimental procedures were approved by the University of Oxford ethical review committee and licensed by the Home Office under the Animal (Scientific procedures) Act 1986. No statistical method was used to predetermine sample size. The experiments were not randomised and the investigators were not blinded to allocation during experiments and outcome assessment.
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