engen spy cas9 recombinant protein  (New England Biolabs)

Journal: bioRxiv

Article Title: TEAD4 regulates apical domain homeostasis and cell-positioning to maintain the trophectoderm lineage during preimplantation mouse embryo development

doi: 10.1101/2025.11.18.689016

Figure Lengend Snippet: A) Example single-z-section confocal images of E3.5 blastocysts containing H2B-RFP marked clones (red) generated by 2-cell microinjections with either siNTC (left) or si Tead4 (right). Embryos are IF-stained for KRT8 (green; intermediate filaments) and counterstained with phalloidin (cyan; actin) and DAPI (blue; DNA). Scale bar, 20μm. The number of embryos per group (n) is indicated. Orange arrows denote typical outer-cell KRT8 expression in siNTC embryos, irrespective of clonal origin. White asterisks indicate the absence of detectable KRT8 in outer si Tead4 clones, which can show abscising apical blebs (white arrows). B) KRT8 expression across whole embryos at E3.5 (RFU, normalised), including unmarked and marked clones, in siNTC control (blue) versus Tead4 KD (orange, si Tead4 ) groups. Median values are shown by dashed lines. Significant differences: *p<0.05. C) KRT8 expression in single outer cells (RFU) of siNTC control and si Tead4 blastocysts; data separated for unmarked (grey) and marked (red) clones. Medians (dashed lines) and significant differences (*p<0.05) are indicated. Sample sizes (n) are shown. D) Schematic of the microinjection procedure to generate E3.5 early-blastocysts with control (siNTC) or Tead4 KD (si Tead4 ) marked clones, co-expressing recombinant Krt8 (with or without an N-terminal HA tag) and Krt18 mRNA (50% of cells) to test for phenotypic rescue of Tead4 KD-induced clonal outer/TE-to-ICM cell allocation phenotypes. E) As in A) but for rescue experiments described in D); phalloidin actin staining is replaced by TEAD4 IF (cyan) to confirm specific KD (white asterisks). Yellow asterisks indicate enhanced recombinant KRT8 expression and intermediate filament formation in marked inner-cell clones (of both treatment groups, atypical of endogenous expression). Blue arrows highlight abscised apical bleb-derived surface vesicles from outer Tead4 KD clones (recombinant KRT8 expression data expanded in Fig. S13). F) Outer/TE and ICM cell-number contributions of marked siNTC versus si Tead4 clones expressing recombinant Krt8 and Krt18 mRNAs. Medians (dashed lines); significant differences: *p<0.05. Note that Krt8 / Krt18 overexpression did not rescue Tead4 KD-induced outer/TE-to-ICM cell allocations (expanded in Fig. S13E–F). G) Schematic of parallel microinjections to generate E3.5 early-blastocysts with control (siNTC) or Tead4 KD (si Tead4 ) marked clones (50% of cells), co-injecting either control dsEGFP or ds Rnd1 plus ds Rnd3 constructs to attempt phenotypic rescue. H) As in E) but for the rescue experiments described in G); TEAD4 IF (cyan) replaces KRT8 staining. White asterisks denote outer-marked si Tead4 clones, some with disrupted apical domains (white arrows). I) Outer/TE and ICM cell-number contributions for marked siNTC/dsEGFP (blue) versus si Tead4 /ds Rnd1 & ds Rnd3 (orange) clones. Medians (dashed lines); significant differences: *p<0.05. Previously significant Tead4 KD–induced increases in ICM contribution are rescued by concomitant Rnd1 / Rnd3 KD, but apical-domain distortions persist, indicating partial phenotypic rescue (expanded in Fig. S14A). Quantified KRT8 expression values (RFU) for panels B) and C) are summarised in S.Tabs. 17; raw clonal-allocation data are in S.Tabs. 19 (Krt8 & Krt18 over-expression; as in panel F) and S.Tabs. 20 (Rnd1 & Rnd3 KD; as in panel I). Statistical analyses were performed based on determined data distribution (normal or non-normal) and application of appropriate tests; details in supplementary statistics Excel workbook .

Article Snippet: For CRISPR-Cas9 targeting of Cdx2 , recombinant poly-adenylated Cas9 mRNA (in this case, Cas9 fused to monomeric streptavidin; Cas9-mSA ) was synthesised from NotI -linearised pCS2+Cas9-mSA plasmid (Addgene #103882) template, using the mMESSAGE mMACHINE SP6 kit (ThermoFisher).

Techniques: Clone Assay, Generated, Staining, Expressing, Control, Microinjection, Recombinant, Derivative Assay, Over Expression, Construct