ythdf3 knockout murine esc lines  (Addgene inc)

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Mettl3 is essential for female mice fertility. (A). Phylogenetic tree of the protein sequences of Ythdf1, Ythdf2 and Ythdf3, based on UCSC database. The three readers appeared together in vertebrates, possibly after whole genome duplication. (B) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in ICR wild-type (WT) oocytes. (BF) Bright field. (C) H&E staining of ovaries, showing normal follicle structure in Mettl3f/f VasaCre− control, and a severe abnormality in Mettl3f/f VasaCre+ females. (D) Gross morphology of Mettl3f/fVasaCre+ and control female ovaries. Cre+ females show a smooth shape that lacks the typical follicular morphology. (E) Number of pups per plug produced by mating Mettl3f/f Vasa Cre+ females, compared with Mettl3f/f Vasa Cre− control females. The fathers in both cases were WT. A significant difference between Cre+ and Cre− female fertility is observed (P < 0.0001, Mann–Whitney test). (F) H&E staining of ovaries, showing normal morphology both in Mettl3f/f Zp3 Cre− and Mettl3f/f Zp3 Cre+ ovaries. (G) Number of pups per plug produced by mating a Mettl3f/f Zp3 Cre+ female, compared with a Mettl3f/+ Zp3 Cre+ control female. The fathers in both cases were WT. A significant difference between f/f and f/+ female fertility is observed (P < 0.0001, Mann-Whitney test). (H) Number of oocytes per mouse flushed from Mettl3f/f Zp3 Cre+ females, compared with Mettl3f/f Zp3 Cre− control females. A significant difference between the number of oocytes of Mettl3f/f ZP3 Cre+ and Mettl3f/f ZP3 Cre− is observed (P < 0.0002, Mann–Whitney test). (I, top) Experimental design – Mett3f/f Zp3 Cre+ and Cre− as control underwent hormone priming, oocyte flushing, fixation, and staining for tubulin. (Bottom) Number of oocytes observed in the different stages of meiosis. In the control, most of the oocytes were in MI stage, in KO (Cre+) all of the observed oocytes were in the GV state. (J) Staining examples of oocytes in the different stages of meiosis as observed in KO (Cre+) and control (Cre−). (K) Differentially expressed genes between Mettl3f/f Zp3 Cre− (control) and Mettl3f/f Zp3 Cre+ (KO) oocytes, along with selected enriched categories. m6A methylated genes appear in bold. Ninety-six genes are up-regulated in the KO, and 117 are down-regulated in the KO.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Immunostaining, Staining, Produced, MANN-WHITNEY, Methylation

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Characterization of compound Ythdf1/2/3 KO mice. (A, left) Statistics of KO offspring received from crossing of Heterozygous mice from each of the indicated strains (Ythdf1+/−, Ythdf2+/−, and Ythdf3+/−). (Right) Distribution of Ythdf2 WT, HET, and KO offspring at days E13.5, 2 d postnatal (DPN), and 30 DPN (compared with expected ratios). (B) Crossing strategy for generating triple-heterozygous mice that were further crossed, and their offspring statistics are presented in C. (C) Percentage of genotypes received by crossing triple-heterozygous mice, out of 200 pups tested 30 DPN. (Red) Observed percentage; (gray) expected under null assumption. No pups with Ythdf2-KO genotype survived 30 DPN. In Ythdf2+/− genotype, pups with KO in either Ythdf1 or Ythdf3 were born in a sub-Mendelian ratio. χ2 test P-values are indicated. (D) Using tetraploid complementation assay, triple-KO embryos were generated and examined on E7.5. (Bottom) H&E staining showing aberrant morphology of triple-KO E7.5 embryos, compared with WT control.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Generated, Staining

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf2 is essential for male mice fertility. (A) H&E staining showing mild degenerative changes, including scattered vacuoles marked by arrows in the seminiferous tubules in Ythdf2-KO males, compared with WT control. (B) Schematic representation of spermatogenesis inside seminiferous tubules. Differentiation is progressing from spermatogonia at the periphery, via spermatocytes and round spermatids, and ends with elongated mature sperm in the center of the tubule. (C, left) H&E staining of the cauda epididymis, showing severe loss of sperm in Ythdf2-KO compared with control. (Right) Bright field of sperm extracted from the cauda epididymis of KO and control, showing a severe reduction in normal sperm quantity in the KO sample, compared with control. (D) Number of pups per plug produced by mating Ythdf2−/− males, compared with Ythdf2+/− control males. The mothers in both cases were WT. A significant difference between the fertility of KO and heterozygous males is observed (P < 0.0001, Mann–Whitney test). (E) Immunostaining of Ythdf1, Ythdf2, Ythdf3, and Mettl3 in seminiferous tubules, showing that each of the Ythdf proteins is expressed at different stages of spermatogenesis. Costaining of Gata4 typically marks Sertoli cells, costaining of γ-H2AX marks different cells during early spermatogenesis. (F) Dimension reduction representation of single-cell RNA-seq (UMAP) measured in adult mouse testis, showing mild expression of Ythdf1 and Ythdf3 in spermatogonia and in Sertoli cells, and more substantial expression of Ythdf2 in spermatogonia and in spermatocytes.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Staining, Produced, MANN-WHITNEY, Immunostaining, RNA Sequencing Assay, Expressing

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf1, Ythdf2, and Ythdf3 are redundant in mouse naïve ESC maintenance. (A) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in WT mESCs, showing a protein expression in the cytosolic compartment of the cell. (B) Cell growth curve of all KO lines and WT control. Cells were grown on mouse feeders, in serum/LIF conditions. (C) Bright field and immunostaining of Nanog (green), Esrrb (red), and DAPI (blue) in KO cells (single, triple, and Mettl3) and WT control, showing that all cell lines express Nanog and Esrrb.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Immunostaining, Expressing

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf1, Ythdf2, and Ythdf3 are functionally redundant in ESC differentiation. (A) Teratomas generated from KO cell line and from WT control. Single-KO teratomas show all germ layers, while triple-KO (TKO) teratomas are poorly differentiated. Selected differentiated structures are marked by arrows. (B) Immunostaining of triple-KO and WT control to Oct4 (red), Foxa2 (green), Tuj1(purple) and DAPI (blue). Triple-KO teratomas contain patches of Oct4 staining, unlike WT teratomas. (C) Alkaline phosphatase (AP) staining of disassociated teratomas from triple-KO and WT control samples, showing a greater AP staining in the Ythdf triple-KO. (D) RT-PCR of pluripotency genes (left) and differentiation genes (right), measured in WT mESCs, and mEBs from the following cell lines: WT control, Ythdf single-KO, Ythdf triple-KO, and Ythdf triple-KO + overexpression (OE) of Ythdf1/2/3 (rescued cell lines). Triple-KO EBs express pluripotent markers and repress differentiation markers similarly to mESCs. Rescued EBs express differentiation markers, similarly to single-KO EBs. (E) EBs were induced for 7 d from the indicated cell lines, and dissociated into single cells at day 7 and comparable amounts of cells were replated in ES medium. Undifferentiated AP+ colonies number per 1000 plated cells was evaluated 5 d later.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Generated, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction, Over Expression

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf triple-KO has a dramatic effect on gene expression. (A) Hierarchical clustering of samples based on Pearson correlation, showing that single-KO samples are highly similar to WT. (B) Number of differentially expressed genes in each of the KO cell lines, compared with WT. (Black) Down-regulated genes, (gray) up-regulated genes. (C) RNA-seq and m6A methylation landscape of selected genes. Normalized coverage is presented. Only Nanog and Dnmt3l are m6A methylated. Dnmt3l, Zscan4a, Zscan4b, Zscan4d, and Dppa3 are overexpressed in triple-KO. (D) Enrichment of up-regulated genes in each category, to early embryo genes (Gao et al. 2017). Genes that are up-regulated in KO of Ythdf1 and Ythdf3 are specifically enriched for two-cell embryo genes. (E) Normalized expression of Ythdf1, Ythdf2, and Ythdf3, as measured in early mouse embryo (Gao et al. 2017). (F) CLIP coverage over m6A peaks of Ythdf readers as measured in humans (n = 41,885) (Patil et al. 2016), and in mice (n = 9861), showing high correlation between coverage by different Ythdf readers.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Expressing, RNA Sequencing Assay, Methylation