plx trc311 vector  (Addgene inc)

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: Etv1 expression is most strongly correlated with expression of Gcg , Pyy and Cck in vivo . (A) Workflow for analysis of enteroendocrine cells (EECs) from the single-cell RNA-sequencing (scRNA-seq) dataset published by . PCA, principal component analysis; UMAP, Uniform Manifold Approximation and Projection. (B) Expression of Etv1 across different cell type clusters (original cell type annotation). TA, transit amplifying. (C) UMAP plot following unsupervised clustering of EECs from mouse small intestine. Clusters are annotated based on expression of known marker genes with a temporal expression pattern during EEC differentiation. EC, enterochromaffin cell. (D) Violin plots showing expression of selected genes involved in EEC differentiation across cell type clusters of EECs from mouse small intestine. (E) UMAP plot showing expression levels of Etv1 in EECs from mouse small intestine. (F) Correlation between Etv1 expression and expression of different enteroendocrine (EE) hormones. R-value=Pearson correlation coefficient. (G-I) UMAP plots showing expression of Gcg (G), Pyy (H) and Cck (I) in EECs from mouse small intestine.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Expressing, In Vivo, RNA Sequencing, Marker

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: EEC differentiation and Etv1 expression in organoid cultures resemble in vivo observations. (A) Expression of Etv1 across different cell type clusters (cell type annotation from ). (B) UMAP plot following unsupervised clustering of EECs from organoid cultures. Clusters are annotated based on expression of known marker genes with a temporal expression pattern during EEC differentiation. (C) Violin plots showing expression of selected transcription factors involved in EEC differentiation across cell type clusters of EECs in organoids. (D) UMAP plot showing expression levels of Etv1 in EECs from organoids. (E) Correlation between Etv1 expression and expression of different EE hormones in organoid cultures. R-value=Pearson correlation coefficient.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Expressing, In Vivo, Marker

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: Etv1 mutant cultures have reduced Pyy expression. (A) Strategy for generation of Etv1 mutant organoid lines. Ngn3, Neurog3 . Created in BioRender by Jensen Team (2025). https://BioRender.com/eqtfeop . This figure was sublicensed under CC-BY 4.0 terms. (B) Amplified and sequenced Etv1 cDNA aligned to the Etv1 gene using the BLAT alignment tool. Screenshot downloaded from http://genome.ucsc.edu . (C) ETV1 protein (transcript variant 1). One dot corresponds to one amino acid (AA). Skipping of exon 8 changes AA 186-187 from phenylalanine (F) and arginine (R) to serine (S) and alanine (A) and introduces a premature stop codon after AA187, resulting in a protein that lacks the DNA binding domain (orange dots). (D-G) Expression of Etv1 (D), Gcg (E), Cck (F) and Pyy (G) in control and Etv1 mutant organoid cultures. The Etv1 reverse primer is located within exon 8. Expression is normalised to expression of Gapdh. Error bars indicate s.d. ( n =3). Significance was evaluated with an unpaired two-tailed t -test. CTRL, control. (H,I) Percentage of Neurog3-RFP + (H) and Gcg-Venus + (I) cells in control (two lines) and Etv1 mutant (three lines) organoid cultures assessed by flow cytometry. Error bars indicate s.d. Significance was evaluated with an unpaired two-tailed t -test. (J,K) Expression of Etv1 (J) and Pyy (K) in control and Etv1 mutant organoid cultures treated for 3 days with or without 10 µg DAPT and/or 20 ng/ml BMP-4. Significance was evaluated with an unpaired two-tailed t -test. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Mutagenesis, Expressing, Amplification, Variant Assay, Binding Assay, Control, Two Tailed Test, Flow Cytometry

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: Etv1 mutant organoids show no overall changes in cell type composition but lack EECs with high Pyy expression. (A) UMAP plot of cells from both control (two lines) and Etv1 mutant (three lines) organoids following scRNA-seq (1158 cells in total). Cell types are annotated based on expression of known marker genes ( Fig. S4A ). (B) UMAP plot of cells from control (left) and Etv1 mutant (right) organoids (control, 494 cells; Etv1 mutant, 664 cells). (C) Percentage of cells found in each of the identified cell clusters in control (two lines) and Etv1 mutant (three lines) organoids. Error bars indicate s.d. Significance was evaluated with an unpaired two-tailed t -test. ns, not significant. (D) Violin plots showing expression levels of known cell type and proliferation marker genes in control (orange) and Etv1 mutant (green) organoids. (E) UMAP plot showing EECs in control (orange) and Etv1 mutant (green) organoids. (F) UMAP plot showing expression levels of Etv1 , Gcg , Cck and Pyy in EECs from control (top row) and Etv1 mutant (bottom row) organoids.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Mutagenesis, Expressing, Control, Marker, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: Etv1 overexpression increases expression of Pyy and Cck , but not Gcg . (A) Strategy for generation of Etv1 -overexpressing ( Etv1 OE) organoids. SI, small intestine. Created in BioRender by Jensen Team (2025). https://BioRender.com/eqtfeop . This figure was sublicensed under CC-BY 4.0 terms. (B) Images of control and Etv1 OE organoids with and without 48 h of doxycycline treatment. Scale bars: 275 µm. Organoids were derived from a Neurog3 -RFP; Gcg -Venus mouse ( ; ). (C) Expression of Etv1 , Pyy , Gcg , Cck , Sct and Ngn3 ( Neurog3 ) in control and Etv1 OE organoid cultures with and without 48 h doxycycline treatment. Error bars indicate s.d. ( n =3). Expression is normalised to expression of 36B4 ( Rplp0 ). Significance was evaluated with a one-way ANOVA. (D) Luciferase activity in inducible Etv1 OE HEK293 cells transfected with a pGL4.23 vector containing either a wild-type (PyyProm_WT) or mutated (PyyProm_MUT) version of a 517 bp region upstream of Pyy covering two putative ETV1 binding sites ( Fig. S7A ). Luciferase activity was normalised to the activity in HEK293 cells transfected with a pGL4.23 vector without any insert. Where indicated, cells were treated for 24 h with doxycycline (1 mg/ml). Error bars indicate s.d. ( n =4). Significance was evaluated with an unpaired two-tailed t -test. ns, not significant; * P <0.05, ** P <0.01.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Over Expression, Expressing, Control, Derivative Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Two Tailed Test