tol2 transgenic vector e1b egfp tol2  (Addgene inc)

Journal: bioRxiv

Article Title: Hand2 represses non-cardiac cell fates through chromatin remodeling at cis- regulatory elements

doi: 10.1101/2023.09.23.559156

Figure Lengend Snippet: (A) Schematic representation of the hand2enh reporter construct. Construct contains a putative enhancer 28Kb upstream of the hand2 TSS (blue rectangle; hand2enh ), an e1b minimal promoter (orange) and eGFP (green), flanked by minimal Tol2 cis sequences to permit integration by Tol2 transposase. (B) Confocal images of reporter expression in the LPM of a 14hpf Tg(hand2enh:eGFP ) embryo (dorsal view, anterior to the top), and in the heart of a 48 hpf Tg(hand2enh:eGFP) ; Tg(myl7:cherry-Ras) embryo (ventral view). (C) Depiction of the Danio rerio hand2 locus with the CRISPR-Cas9 target site used to generate hand2 crispants at 260bp into exon 1, marked with a yellow lightning bolt. PCR primers used for fragment analysis to confirm CRISPR-induced mutagenesis are shown as red arrowheads. (D) Epifluorescence images of Tg(hand2enh:eGFP) embryos at 14 (dorsal view, anterior to the top) and 48 hpf (lateral view, anterior to the left): wildtype (WT), hand2 mutant ( hanS6 ), and hand2 crispant ( hand2 -CRP). White arrows indicate residue myocardial tissue in hand2 mutant and crispant. (E) Representative capillary electrophoresis traces of WT and hand2 -CRP embryos, plotted as the number of bases from the start of the amplified fragment. The percentage of cells with indel mutations for 9 representative embryos is indicated at right. (F) Confocal images of control (uninjected) and crispant Tg(myl7:nucGFP) embryos at 20-ss (19 hpf), ventral view. hand2 crispants have significantly fewer myocardial cells than wildtype embryos (unpaired two-tailed t-test; *p < 0.0001). (G) Confocal images, ventral view, of whole mount Tg(myl7:nucGFP) embryos at 19 hpf. WT and hand2 crispant transgenic embryos, immunostained with an antibody against aPKCs (purple) and nuclear-counterstained with DAPI (cyan); myocardial cells are labeled by myl7-driven nuclear GFP expression (green). Scale bars: 50um in B, D; 30um in F; 20um in G.

Article Snippet: Upstream cis -regulatory elements were amplified from wildtype zebrafish genomic DNA and cloned into the e1b-eGFP-Tol2 vector (available from Addgene, Plasmid #37845) by restriction cloning at the XhoI and BglII sites.

Techniques: Construct, Expressing, CRISPR, Mutagenesis, Residue, Electrophoresis, Amplification, Control, Two Tailed Test, Transgenic Assay, Labeling

Journal: bioRxiv

Article Title: Hand2 represses non-cardiac cell fates through chromatin remodeling at cis- regulatory elements

doi: 10.1101/2023.09.23.559156

Figure Lengend Snippet: (A) Confocal images of wildtype and hanS6 14 hpf embryos injected with the eDAR1031-e1b-eGFP reporter construct, showing reporter gene expression in the LPM (yellow dashed region) only in the hand2 mutant embryo (lower panel). Scale bars are 50um. (B) Quantification of wildtype and hanS6 embryos injected with eDAR1031-e1b-eGFP that exhibit eGFP expression within the LPM. “N” denotes the number of independent experimental replicate. “n” denotes the total number of embryos quantified. eGFP expression from embryos injected with the e1b-eGFP construct are shown as a control (*, p-value < 0.05, ns = not significant). (C) Putative Hand2-regulated gene network compiled based on the analysis of genes that are downregulated (blue text) or upregulated (red text) in the absence of Hand2. Lines with arrowheads signify an activator function for Hand2 in the indicated biological process while lines with a block arrow signify a repressive function for Hand2 in the indicated biological process. Dashed lines indicate Hand2 may either directly or indirectly regulate list genes. (D) Proposed mechanism of Hand2-dependent regulation of non-LPM and LPM-derived tissues based on the results of this study. Schematic of a 14 hpf zebrafish embryo is shown in the middle with the LPM colored in green. The embryo proper is colored blue while the yolk is colored beige. In the LPM, Hand2 positively regulate expression of LPM genes and limit the accessibility of non-LPM genes. In non-LPM tissues where Hand2 is not expressed, enhancers of non-LPM genes are accessible and can act on target non-LPM genes.

Article Snippet: Upstream cis -regulatory elements were amplified from wildtype zebrafish genomic DNA and cloned into the e1b-eGFP-Tol2 vector (available from Addgene, Plasmid #37845) by restriction cloning at the XhoI and BglII sites.

Techniques: Injection, Construct, Gene Expression, Mutagenesis, Expressing, Control, Blocking Assay, Derivative Assay