Journal: medRxiv

Article Title: Identifying risk genes for embryo aneuploidy using ultra-low coverage whole-genome sequencing

doi: 10.1101/2023.07.22.23292618

Figure Lengend Snippet: (A) Expression and localization of CCDC66 in different stages of mouse oocyte meiotic maturation. Oocytes were stained with anti-CCDC66 antibody (gray) at (0, 5, 7, and 16) hours of maturation corresponding to Prophase I, Pro-metaphase I, Metaphase I and Metaphase II, respectively. Tubulin and DAPI was used to label the spindle and DNA (green and blue, respectively). (B) Prophase-I arrested oocytes were co-microinjected with Trim21 cRNA and either CCDC66 antibody or IgG. Oocytes were fixed at metaphase I and stained to detect CCDC66 (gray). Tubulin (green) and DAPI (blue) were used to label the spindle and DNA. (C) Relative CCDC66 intensity from B, two-tailed unpaired Students t-Test (***p < 0.001). (D) Quantification of percentage of polar body extrusion (PBE). (E) Percentage of aneuploid Metaphase II eggs after knockdown of CCDC66 in young and old mouse oocytes (One-way ANOVA, * p <0.05; ** p < 0.01, *** p < 0.001). Number of oocytes examined: young IgG: 34, young CCDC66: 37, old IgG: 29, old CCDC66: 31. These experiments were repeated 3 times. Scale bars: 15 and 4 μm (insets).

Article Snippet: Rabbit anti-CCDC66 antibody (Bethyl Laboratories, #A303-339A) and control IgG antibody (Merck Millipore, #12-370) were purified using Amicon Ultra 0.5-ml Centrifugal Filter (Merk Millipore, #UFC5003096). pGEMHE-Cherry-TRIM21 (Addgene, #105522) or pGEMHE-mEGFP-mTrim21 (Addgene, #105519) were linearized with Asc I (New England Biolabs, #R0558S,) and in vitro transcribed using a T7 mMessage mMachine Kit (Ambion, #AM1340).

Techniques: Expressing, Staining, Two Tailed Test

Journal: medRxiv

Article Title: Identifying risk genes for embryo aneuploidy using ultra-low coverage whole-genome sequencing

doi: 10.1101/2023.07.22.23292618

Figure Lengend Snippet: For each IVF cycle, the number of aneuploidy and euploidy embryo biopsies were determined using PGT-A. ulc-WGS data from PGT-A for each IVF cycle were combined for analysis. Genotype likelihood and dosage were imputed for each variant and ancestry of the samples was inferred using the genotype likelihood. Association tests were performed between the imputed genetic variants and the aneuploidy rate. eQTL analysis was used to determine the candidate gene associated with the top variants from the association test. CCDC66 was selected for functional studies using a mouse oocyte model.

Article Snippet: Rabbit anti-CCDC66 antibody (Bethyl Laboratories, #A303-339A) and control IgG antibody (Merck Millipore, #12-370) were purified using Amicon Ultra 0.5-ml Centrifugal Filter (Merk Millipore, #UFC5003096). pGEMHE-Cherry-TRIM21 (Addgene, #105522) or pGEMHE-mEGFP-mTrim21 (Addgene, #105519) were linearized with Asc I (New England Biolabs, #R0558S,) and in vitro transcribed using a T7 mMessage mMachine Kit (Ambion, #AM1340).

Techniques: Variant Assay, Functional Assay

Journal: medRxiv

Article Title: Identifying risk genes for embryo aneuploidy using ultra-low coverage whole-genome sequencing

doi: 10.1101/2023.07.22.23292618

Figure Lengend Snippet: (A) GTEx eQTL of the lead significant variants rs12495172 (chr3-55959628-G-A) on CCDC66 expression. The decreased expression of CCDC66 correlates with alternative alleles of rs12495172. (B) The genotype dosages of the alternative allele of rs12495172. The alternative allele genotype dosages in samples were divided into 3 bins of roughly equal size. The aneuploidy rates among the samples were positively correlated with the alterative allele genotype dosages.

Article Snippet: Rabbit anti-CCDC66 antibody (Bethyl Laboratories, #A303-339A) and control IgG antibody (Merck Millipore, #12-370) were purified using Amicon Ultra 0.5-ml Centrifugal Filter (Merk Millipore, #UFC5003096). pGEMHE-Cherry-TRIM21 (Addgene, #105522) or pGEMHE-mEGFP-mTrim21 (Addgene, #105519) were linearized with Asc I (New England Biolabs, #R0558S,) and in vitro transcribed using a T7 mMessage mMachine Kit (Ambion, #AM1340).

Techniques: Expressing

Journal: medRxiv

Article Title: Identifying risk genes for embryo aneuploidy using ultra-low coverage whole-genome sequencing

doi: 10.1101/2023.07.22.23292618

Figure Lengend Snippet: (A) Expression and localization of CCDC66 in different stages of mouse oocyte meiotic maturation. Oocytes were stained with anti-CCDC66 antibody (gray) at (0, 5, 7, and 16) hours of maturation corresponding to Prophase I, Pro-metaphase I, Metaphase I and Metaphase II, respectively. Tubulin and DAPI was used to label the spindle and DNA (green and blue, respectively). (B) Prophase-I arrested oocytes were co-microinjected with Trim21 cRNA and either CCDC66 antibody or IgG. Oocytes were fixed at metaphase I and stained to detect CCDC66 (gray). Tubulin (green) and DAPI (blue) were used to label the spindle and DNA. (C) Relative CCDC66 intensity from B, two-tailed unpaired Students t-Test (***p < 0.001). (D) Quantification of percentage of polar body extrusion (PBE). (E) Percentage of aneuploid Metaphase II eggs after knockdown of CCDC66 in young and old mouse oocytes (One-way ANOVA, * p <0.05; ** p < 0.01, *** p < 0.001). Number of oocytes examined: young IgG: 34, young CCDC66: 37, old IgG: 29, old CCDC66: 31. These experiments were repeated 3 times. Scale bars: 15 and 4 μm (insets).

Article Snippet: Rabbit anti-CCDC66 antibody (Bethyl Laboratories, #A303-339A) and control IgG antibody (Merck Millipore, #12-370) were purified using Amicon Ultra 0.5-ml Centrifugal Filter (Merk Millipore, #UFC5003096). pGEMHE-Cherry-TRIM21 (Addgene, #105522) or pGEMHE-mEGFP-mTrim21 (Addgene, #105519) were linearized with Asc I (New England Biolabs, #R0558S,) and in vitro transcribed using a T7 mMessage mMachine Kit (Ambion, #AM1340).

Techniques: Expressing, Staining, Two Tailed Test