eb3 tdtomato  (Addgene inc)

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) Mouse homeobox genes that are specifically expressed during ZGA. The genes were identified by means of statistically determined k -means clustering based on their expression in preimplantation embryos. Dux is shown in a bold italic font. ( B ) Scatterplot showing per-gene normalized read counts in mouse totipotent totipotent blastomere-like cells (TBLCs) versus mouse embryonic stem cells (mESCs). Genes with more than tenfold normalized read counts (FC = 10) have been highlighted. Dux is shown in a bold italic font. ( C ) MA plot displaying gene expression in Dux knockout 2C-embryos versus WT 2C-embryos. Loss of Dux partially downregulates the multicopy homeobox gene Obox4 . ( D ) Immunofluorescence staining of OBOX4 and MERVL GAG at different preimplantation embryo stages. ( E ) Immunofluorescence staining of DUX, OBOX4, and OCT4 in 2C-like mESCs. ( F ) Translation profile of DUX and OBOX4 during preimplantation characterized by Ribo-seq signal.

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Expressing, Gene Expression, Knock-Out, Immunofluorescence, Staining

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) Expression profiles of k -means clustered gene sets during preimplantation development based on z -scored expression of scRNA-seq and bulk RNA-seq. Genes appeared in both scRNA-seq cluster 1 and bulk RNA-seq cluster 4 were designated as 2C-genes. ( B ) Western blot showing protein products of the candidate genes ectopically expressed in mouse embryonic stem cells (mESCs) at 18 hr post transfection. ( C ) Bar plot showing MERVL transcript level in mESCs at 18 hr post Dux or Obox4 transfection, detected by reverse transcription quantitative real-time PCR (RT-qPCR). n = 3 biological replicates. Error bars indicate standard deviations. Figure 2—figure supplement 1—source data 1. Original and uncropped blot for .

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Expressing, RNA Sequencing, Western Blot, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) Diagram of the 2C::tdTomato reporter assay. mESCs bearing the tdTomato expression cassette under the control of the MERVL long terminal repeat (LTR) promoter showed red fluorescence upon entering the 2C-like state. The expression of the transcription factor increased the 2C-like population, as detected using FACS. An EGFP expression plasmid was co-transfected with a gene of interest to normalize the transfection efficiency. ( B ) Boxplot showing normalized 2C-like cell percentage in 2C::tdTomato reporter mESCs overexpressing candidate pioneer factors. Dux and Obox4 potently induced a 2C-like state. n = 3 biological replicates. Error bars indicate standard deviations. ( C ) Upper panel: schematic of Obox4 -inducible cell line construction. Lower panel: western blot showing OBOX4 level upon induction by different concentrations of doxycycline. Expression of OBOX4 was carried out in a dose-dependent manner. ( D ) Left panel: volcano plot of differentially expressed genes (DEGs) in mESCs with Obox4 induction for 48 hr. Representative 2C-genes are labeled with gene symbols. Right panel: expression profile of genes upregulated by Obox4 during embryogenesis. n = 3 biological replicates. ( E ) Left panel: volcano plot of differentially expressed transposable elements in mESCs with Obox4 induction for 48 hr. MERVL and MERVK elements were highlighted. Right panel: expression profile of MERVL and MERVK elements during preimplantation embryogenesis. n = 3 biological replicates. ( F ) Heatmaps of the expression of 2C-genes in preimplantation embryos, naturally occurred 2C-like mESCs, and induced 2C-like mESCs. ( G ) Venn diagram showing overlap of 2C-genes with genes induced by ectopic expression of Dux and Obox4 in mESCs. ( H ) Scatterplot showing per-gene expression changes in Dux -induced versus Obox4 -induced mESCs. 2C-genes are highlighted in red. Figure 2—source data 1. Original and uncropped blot for .

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Reporter Assay, Expressing, Control, Fluorescence, Plasmid Preparation, Transfection, Western Blot, Labeling, Gene Expression

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) FACS analysis of 2C::tdTomato cells co-transfected with candidate gene expression plasmids and EGFP. ( B ) Fluorescence microscopy of 2C::tdTomato mESCs transfected with empty plasmid, plasmid encoding DUX, or plasmid encoding OBOX4. Plasmids encoding EGFP were co-transfected to normalize the transfection efficiency in FACS analysis.

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Transfection, Gene Expression, Fluorescence, Microscopy, Plasmid Preparation

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) Schematic diagram of the cleavage under targets and release using nuclease (CUT&RUN) workflow using mouse embryonic stem cells (mESCs) with induced expression of FLAG-tagged DUX. mESCs bearing a doxycycline-inducible 3×FLAG-DUX expression cassette were induced with doxycycline for 24 hr. DUX-associated DNA was pulled down in the CUT&RUN assay using a high-affinity anti-FLAG antibody. ( B ) Left panel: comparison of DUX binding motif predicted with published ChIP data and CUT&RUN result from this study. Right panel: overlap of DUX binding peaks discovered with the two data sets. ( C ) Heatmaps showing the OBOX4 binding site distribution near top covered long terminal repeat (LTR) elements. ( D ) Expression profile of top 5 OBOX4 covered LTR elements during embryogenesis.

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Expressing, Comparison, Binding Assay

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) Left panel: pie chart displaying proportions of annotated genomic regions of the OBOX4 binding sites. Right panel: heatmap showing the OBOX4 binding site distribution near 2C-gene promoters. ( B ) Left panel: the predicted OBOX4 binding motif using the top 500 cleavage under targets and release using nuclease (CUT&RUN) peaks. Right panel: histograph showing the distribution of the predicted OBOX4 binding motif near 2C and random gene promoters. ( C ) Venn diagram showing the distinct and overlapping 2C-genes targeted by DUX and OBOX4. ( D ) Representative genomic track showing DUX and OBOX4 binding sites at the Dppa2 , Sp110, and Zscan4d loci and their expression levels in Obox4 and Dux -induced mouse embryonic stem cells (mESCs). Read counts were CPM normalized. The OBOX4 binding sites overlapped with those of DUX. Dppa2, Sp110 , and Zscan4d expression was upregulated upon Obox4 and Dux induction. ( E ) Left panel: pie- hart displaying proportions of annotated transposable elements (TEs) of the OBOX4 binding sites. Right panel: bar plot showing the top 10 OBOX4 covered LTR elements. ( F ) Left panel: pie chart displaying proportions of annotated TEs of the DUX binding sites. Right panel: bar plot showing the top 10 DUX covered LTR elements. ( G ) Representative genomic track showing DUX and OBOX4 binding sites at MT2_Mm and RLTR13B2. Read counts were CPM normalized. The OBOX4 binding sites overlapped with those of DUX at MT2_Mm loci, while RLTR13B2 was uniquely bound by OBOX4. ( H ) Schematic design of the LTR::tdTomato reporter assay. Plasmids bearing a tdTomato ORF downstream of MT2_Mm or RLTR13B2 were co-transfected with Dux or Obox4 expression plasmids. Activation of LTR elements resulted in an increased red fluorescence-positive mESC population, which was measured using FACS. EGFP expression plasmid was co-transfected into the culture, following which the green fluorescence-positive population was measured, to normalize the transfection efficiency. ( I ) Bar plots showing the percentage of red fluorescence-positive mESCs upon expression of Dux or Obox4 . Both Dux and Obox4 activated MT2_Mm, whereas only Obox4 activated RLTR13B2.

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Binding Assay, Expressing, Reporter Assay, Transfection, Activation Assay, Fluorescence, Plasmid Preparation

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) Agarose gel image of PCR-based genotyping of the Obox4 knockout allele in founder mouse embryonic stem cell (mESC) lines. The red arrows indicate cell lines bearing biallelic deletions of the Obox4 cluster. ( B ) Agarose gel image of PCR-based genotyping of the Dux knockout allele in founder mESC lines. The red arrows indicate cell lines without the genomic Dux sequences. ( C ) Bar plot showing copy number of Obox4 in the founder mESC lines analyzed in ( A , B ), as detected by qPCR using purified genomic DNA. Figure 3—figure supplement 3—source data 1. Original and uncropped gels for . Figure 3—figure supplement 3—source data 2. Original and uncropped gels for .

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Agarose Gel Electrophoresis, Knock-Out, Purification

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) Schematic representation of the somatic cell nuclear transfer (SCNT) experiment. The nuclei of knockout mouse embryonic stem cells (mESCs) were transferred into enucleated oocytes to generate zygotes with knockout genotype. ( B ) Upper panel: percent SCNT embryos developed to different stages at 4 days post nuclear transfer (dpt). Lower panel: p-value of non-parametric ANOVA among different genotypes and stages compared to wildtype SCNT embryos. Three independent experiments were conducted, with 150–200 embryos per condition. ( C ) Representative picture of SCNT embryos at 4 dpt. Morphologically abnormal blastocysts are highlighted. Blastocysts generated by double knockout mESCs were severely defective. ( D ) Schematic representation of CRISPR-Cas9-mediated Dux and Obox4 knockout mouse production. In vitro fertilized mouse zygotes were electroporated with pre-assembled CRISPR-Cas9 complex targeting Dux and Obox4 loci. ( E ) Bar plot showing genotype percentage of the pups delivered by Obox4 Het intercrosses. Four litters delivered 29 pups, litter size 7.25 ± 1.26. ns p-value=0.9146, chi-square goodness-of-fit test. ( F ) Representative photos of Obox4 KO and WT adult mice analyzed in ( E ). ( G ) Photo of Obox4 KO intercross litter with live pups. ( H ) Bar plot showing genotype of Obox4 allele in the pups delivered by crossing of Dux KO / Obox4 Het × Dux KO / Obox4 Het or Dux KO / Obox4 Het × Dux Het / Obox4 Het . Nine litters delivered 54 pups, Dux heterozygous and knockout allele were present in 31 and 23 pups, respectively. **p-value=0.001306; *p-value=0.02218; chi-square goodness-of-fit test. ( I ) Bar plot showing observed percentages of different preimplantation stage embryos bearing Obox4 KO allele with Dux heterozygous or knockout allele at 4.5 days post coitum (dpc). Among the total 94 embryos assessed, 2 degenerated, 10 two-cell arrest, 2 4–8-cell arrest, 4 morula arrest embryos were observed at 4.5 dpc, whereas 76 developed to blastocyst. ***p-value=3.564 × 10 –5 ; for two-cell; * p-value=0.04348; for blastocyst *p-value=0.02549; chi-square goodness-of-fit test. ( J ) Scatterplot showing expression log 2 fold changes of genes (left panel) and transposable elements (TEs) (right panel) in Dux KO / Obox4 KO and Dux KO / Obox4 Het versus Dux KO / Obox4 WT 2C embryos. 2C-genes and ERVK elements targeted by OBOX4 are highlighted in blue. MT2_Mm and MERVL-int are labeled. In total, 79 and 5 2C-genes were downregulated in Dux KO / Obox4 KO and Dux KO / Obox4 Het 2C embryos, respectively. ( K ) Upper panel: schematic representation of Dux and Obox4 knockdown experiments in preimplantation embryos. Male pronuclei of zygotes were microinjected with antisense oligonucleotide (ASO) targeting Dux or Obox4 transcripts. Lower panel: the percentages of embryonic stages observed at 1.5 dpc, 2.5 dpc, 3.5 dpc, and 4.5 dpc. ( L ) Representative picture of KD embryos at 4.5 dpc. No blastocyst was observed among double ASO knockdown embryos.

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Knock-Out, Generated, Double Knockout, CRISPR, In Vitro, Expressing, Labeling, Knockdown

Journal: eLife

Article Title: Obox4 promotes zygotic genome activation upon loss of Dux

doi: 10.7554/eLife.95856

Figure Lengend Snippet: ( A ) Heatmaps of the expression of 2C-genes in preimplantation embryos, non-induced mouse embryonic stem cells (mESCs), and Dux / Obox4 / Obox3 / Obox5 -induced mESCs. ( B ) Venn diagram showing overlap of 2C-genes induced by ectopic expression of Obox3 , Obox4 , and Obox5 in mESCs. ( C ) Scatterplot showing per-gene expression changes in Obox3 versus Obox5-induced mESCs. 2C-genes are highlighted in red. ( D ) Scatterplot showing per-gene (left panel) and per-transposable element (right panel) expression changes in Obox3 and induced versus Obox4 -induced mESCs. 2C-genes and Obox3 -induced transposable elements (TEs) are highlighted in red. ( E ) Scatterplot showing per-gene (left panel) and per-transposable element (right panel) expression changes in Obox5 and induced versus Obox4 -induced mESCs. 2C-genes and Obox3 -induced TEs are highlighted in red. ( F ) Venn diagram showing overlap of 2C-genes downregulated upon Obox4 knockdown, Dux / Obox4 double knockdown, and Obox maternal-zygotic knockout. ( G ) Scatterplot showing per-gene expression changes in Obox maternal-zygotic knockout versus Dux / Obox4 double knockdown 2C embryos. 2C-genes are highlighted in red. ( H ) Scatterplot showing per-gene expression changes in Obox4 knockdown versus Dux / Obox4 double knockdown (left panel) and Obox maternal-zygotic knockout (right panel) 2C embryos. 2C-genes are highlighted in red. ( I ) Schematic model of different zygotic genome activation (ZGA) strategy employed by human and mouse. Obox family members with redundant functions are expressed in high dose at peri-ZGA stages to ensure rapid ZGA. Obox4 evolved as a divergent Obox family member that provides functional redundancy to Dux .

Article Snippet: The 2C::tdTomato reporter cell line was generated by transfection of EB3 mESCs (Cat# AES0139, RIKEN BRC Cell Bank) with a linearized 2C::tdTomato reporter plasmid (Cat# 40281, Addgene).

Techniques: Expressing, Gene Expression, Knockdown, Knock-Out, Activation Assay, Functional Assay