DEV024539 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Table S10 -

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• Supplemental Table S2 -

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• Supplemental Table S3 -

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• Supplemental Table S4 -

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• Supplemental Table S5 -

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• Supplemental Table S6 -

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• Supplemental Table S7 -

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• Supplemental Table S8 -

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• Supplemental Table S9 -

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• Supplemental Figure S1 -

  Fig. S1. Transcriptional misregulation in CTCF-depleted oocytes. Hierarchical cluster analysis of microarray data. Affymetrix MOE 430 2.0 GeneChips were used to analyze oocyte transcripts from five pairs of non-transgenic and transgenic littermates. Ntg and Tg indicate clustering of non-transgenic and transgenic groups, respectively. Transgenic line 1 is shown.

• Supplemental Figure S2 -

  Fig. S2. Normal patterns of chromatin staining in CTCF-depleted oocytes. Immunofluorescence images of DNA-, (A) dimethylH3K4-, (C) H2Az- and (E) HP1-β-stained GV oocytes. One confocal section of an NSN- or SN-type GV oocyte is shown per panel, with green indicating DNA-stained nuclei and red indicating antibody-stained nuclei. Scale bar: 40 µm. (B) Quantification of nuclear dimethylH3K4 immunofluorescence staining in NSN-type oocytes depicted in A. (D) Quantification of nuclear H2Az immunofluorescence staining in NSN- and SN-type oocytes depicted in C. (F) Quantification of nuclear HP1-β immunofluorescence staining in NSN- and SN-type oocytes depicted in E. n, number of oocytes.

• Supplemental Figure S3 -

  Fig. S3. Triploid CTCF-depleted embryos. Chromosome spreads of embryos. Cleavage-stage embryos were flushed at 72 hours post-hCG, arrested in prometaphase overnight and processed for chromosome spreads. Chromosomes from one embryo are shown in each panel. Boxed chromosomes from the center panel are magnified in the right panel. Eight percent (3/38) of transgenic embryos derived from Line 1 are triploid. n=number of embryos. Scale bar: 0.1 µm.

• Supplemental Figure S4 -

  Fig. S4. Normal chromatid cohesion in CTCF-depleted embryos. (A) Quantification of embryos with resolved sister chromatids after short prometaphase arrest. Two- to four- cell embryos were flushed at 54 hours post-hCG, arrested in prometaphase for 1-2 hours and processed for chromosome spreads. Transgenic line 1 is shown. Examples of unresolved and resolved chromosomes are shown in the two panels below. Scale bar: 0.1 µm. (B) Quantification of embryos with widely separated sister chromatids (open chromosomes) after prolonged prometaphase arrest. Two- to four-cell embryos were flushed at 54 hours post-hCG, arrested in prometaphase for 16 hours and processed for chromosome spreads. Transgenic line 1 is shown. Examples of closed and open chromosomes are shown in the two panels below. Scale bar: 0.1 µm. (C) Immunofluorescence images of DNA, CTCF and SMC1 triple-stained four-cell embryos. Two- to four-cell embryos were flushed at 54 hours post-hCG. One confocal section of an embryo is shown per panel, with green indicating DNA-stained nuclei, red indicating CTCF-stained nuclei and white indicating SMC1-stained nuclei. Transgenic line 1 is shown. All embryos were pooled and pre-extracted prior to fixation and staining. Scale bar: 40 µm. (D) Quantification of nuclear SMC1 immunofluorescence staining in four-cell embryos depicted in C. n, number of embryos.

• Supplemental Figure S5 -

  Fig. S5. Nuclear protein restored by 6 hours after Ctcf mRNA injection. Immunofluorescence images of CTCF-stained embryos after cytoplasmic mRNA microinjection. CTCF-depleted one-cell embryos from Line 1 were microinjected with Ctcf or control Gfp mRNA and cultured for 6 hours. One embryo is shown per panel, with red indicating CTCF-stained nuclei. Scale bar: 40 µm.