Extended Data Fig. 2: CRISPR-Cas based C-terminal tagging of endogenous REC8 does not disrupt accurate oocyte chromosome alignment and segregation.
From: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy
(A) Images from representative time lapse movies of chromosomes (marked with H2B-mScarlet) and meiotic spindles (marked with MAP4 microtubule-binding domain (mNeonGreen-MAP4-MTBD)) in wild-type and REC8-FKBP12F36V-mClover3 oocytes. (B) Representative metaphase II chromosome spreads with centromeres labeled by ACA staining in wild-type and REC8-FKBP12F36V-mClover3 oocytes. (C) The percentage of aneuploid eggs matured in vitro from wild-type or REC8-FKBP12F36V-mClover3 oocytes. (D) Representative images of metaphase II-arrested eggs matured in vitro from REC8-FKBP12F36V-mClover3 oocytes treated with DMSO or dTAG-13 for 1 h and stained with ACA and Hoechst. (E) Quantification of the percentage of eggs with prematurely separated sister chromatids following 1-h dTAG-13 treatment during metaphase I followed by washout and progression into meiosis II. (F) Representative images of embryos at various stages of preimplantation development following fertilization of DMSO- or dTAG-13-treated REC8-FKBP12F36V-mClover3 eggs. (G) Quantification of developmental progression from the zygote to blastocyst stage in embryos generated by fertilization of DMSO- or dTAG-13-treated REC8-FKBP12F36V-mClover3 eggs. Data are from three independent experiments. Statistical significance was assessed using Student’s t-test. The number of analyzed cells or embryos is indicated in brackets and italics.
