recommended standard control morpholino  (Gene Tools Inc)

Journal: bioRxiv

Article Title: H3K4 methylation-promoted transcriptional memory ensures faithful zygotic genome activation and embryonic development

doi: 10.1101/2025.01.20.633863

Figure Lengend Snippet: Cxxc1 and Kmt2b are required for proper ZGA and embryonic development. (A) Gene expression levels of cxxc1.L and cxxc1.S at stages of development (Session et al., 2016). (B) Gene expression levels of kmt2b.L and kmt2b.S at stages of development (Session et al., 2016). (C) Western blots showing Cxxc1 and Kmt2b protein levels in Cxxc1 and Kmt2b knockdown embryos. (D) Count of differentially expressed genes at 5.5hpf, 6.5hpf and 7.5hpf time points in Cxxc1 and Kmt2b morpholino-injected conditions compared to control morpholino embryos (adj. p-value < 0.05). (E) Overlap of downregulated zygotic genes at any timepoint between Cxxc1 and Kmt2b morpholino conditions compared to control morpholino. (F) Overlap of downregulated genes compared to control morpholino at any timepoint between both biological replicates. (G) Number of differentially expressed and unchanged zygotic genes at 5.5hpf, 6.5hpf and 7.5hpf time points in Cxxc1 and Kmt2b morpholino-injected conditions compared to control morpholino embryos in biological replicate 2 (adj. p-value < 0.05). Zygotic genes = Gamete-Zygotic (GZ) + Zygotic-Specific (ZS). (H-I) Fold-change of gene expression levels of all zygotic genes in each group calculated over mean TPM of control morpholino technical replicates for (H) KEPT and (I) GAINED groups at the three time points in biological replicate 2. Statistical test: one-sided Wilcoxon rank-sum test; p-values: (****)<=0.0001, (***)<=0.001, (**) <= 0.01, (*)<=0.05; n.s. are p-values > 0.05. (J) Expression of zygotic genes in 3 technical replicates of every sample, clustered by H3K4me3 dynamics group (KEPT, GAINED and ABSENT) for biological replicate 2. Fold-change of every gene is calculated over mean TPM of three technical replicates of control morpholino of the respective time point. Genes are annotated by differential gene expression, and RNA dynamics group. (K) Gene ontology analysis of downregulated zygotic genes from . (L) Representative phenotype images of embryos for each condition at NF41. n=2 experiments, N>35 per condition.

Article Snippet: The recommended standard control morpholino from Gene Tools LLC was used as a control.

Techniques: Expressing, Western Blot, Knockdown, Injection, Control

Journal: bioRxiv

Article Title: H3K4 methylation-promoted transcriptional memory ensures faithful zygotic genome activation and embryonic development

doi: 10.1101/2025.01.20.633863

Figure Lengend Snippet: Cxxc1 and Kmt2b are required for proper ZGA and embryonic development. (A) Illustration of experimental setup. (B) Western blot showing H3K4me3 levels in Cxxc1 and Kmt2b knockdown embryos compared to non-injected control and control morpholino-injected embryos at post-ZGA (st12). (C) Overlap of downregulated zygotic genes at any timepoint between Cxxc1 and Kmt2b morpholino conditions compared to control morpholino. (D) Volcano plots displaying expression fold-change and adj. p-value (cutoff: 0.05) in knockdown conditions compared to control morpholino at each timepoint colored by RNA dynamics groups. (E) Number of differentially expressed and unchanged zygotic genes at 5.5hpf, 6.5hpf and 7.5hpf time points in Cxxc1 and Kmt2b morpholino-injected conditions compared to control morpholino embryos (adj. p-value < 0.05). Zygotic genes = Gamete-Zygotic (GZ) + Zygotic-Specific (ZS). (F-G) Fold-change of gene expression levels of all zygotic genes in each group calculated over mean TPM of control morpholino technical replicates for (F) KEPT and (G) GAINED groups at the three time points. Statistical test: one-sided Wilcoxon rank-sum test; p-values: (****)<=0.0001, (***)<=0.001, (**) <= 0.01, (*)<=0.05; n.s. are p-values > 0.05. (H) Expression of zygotic genes in 3 technical replicates of every sample, clustered by H3K4me3 dynamics group (KEPT, GAINED and ABSENT). Fold-change of every gene is calculated over the mean TPM of three technical replicates of control morpholino of the respective time point. Genes are annotated by differential gene expression, and RNA dynamics group. (I) Fold change expression of pou5f3.2 and sox3 calculated over mean TPM of control morpholino replicates at two timepoints. (J) Quantification of survival phenotype. n=2 experiments, N>35 embryos per condition.

Article Snippet: The recommended standard control morpholino from Gene Tools LLC was used as a control.

Techniques: Western Blot, Knockdown, Injection, Control, Expressing