addgene plasmid  (Addgene inc)

Journal: Cell Reports Methods

Article Title: African pygmy mouse iPSCs as a model for in vitro embryogenesis, interspecies chimerism, and blastocyst complementation

doi: 10.1016/j.crmeth.2025.101293

Figure Lengend Snippet: Residual contractions of the explanted heart tissue are shown at increased zoom. Nuclear mCHERRY fluorescence signal is visible. The “14x” label is an acquisition software overlay and is not representative of the imaging magnification.

Article Snippet: The T4-gRNA sequence was cloned using Golden Gate Assembly into the PX458 Cas9 backbone plasmid (Addgene #48138) and used together with the previously reported R26-H2B-mCherry (Addgene #137928) or R26-H2B-EGFP (Addgene #137925) HR donor vector for targeting.

Techniques:

Journal: Nature Communications

Article Title: Epidermal growth factor receptor activation is essential for kidney fibrosis development

doi: 10.1038/s41467-023-43226-x

Figure Lengend Snippet: a Schematic of experimental protocol. b Under normal conditions, mCherry-positive cells (both pericytes and fibroblasts) were detected surrounding blood vessels. (representative of 3 studies) Scale bar = 50 μm. c Myofibroblasts isolated from FibEGFR−/− mice after UUO for 7 days expressed markedly lower mRNA levels of Egfr . n = 6. d Marked increases in mCherry+ cells seen in WT mice at day 7 after UUO were significantly attenuated in FibEGFR−/− mice. n = 5. Scale bar = 100 μm. e Single nucleus RNAseq (snRNAseq) analysis identified clusters of kidney cells. f Canonical markers of kidney cell populations were used to identify major cell types in the kidney: podocyte ( Nphs1 ), endothelial cells ( vWF ), proximal tubule (PT) ( Lrp2, slc4a4 ), descending limb of Henle’s loop (DLOH) ( Slc4a11 ), ascending limb of Henle’s loop (ALOH) (Slc12a1), distal convoluted tubules (DCT) ( Slc12a3 ), collecting duct principal cell (CD-PC) ( Slc8a1,Aqp2 ), collecting duct intercalated cell (CD-IC) ( Atp6v1g3 ), pericytes ( Rgs5 ), fibroblast (Fib) ( Dcn,Col1a1 ), myeloid cell ( Cd68,C1qb ) and T/NK cell ( CD3e, Nkg7 ). g Cells with expression of canonical genes for fibroblasts were significantly decreased in FibEGFR−/− mice at day 3 after UUO. h Violin plot indicated less PDGFRβ+ myofibroblasts (MF) in FibEGFR−/− mice (D3 KO) compared to WT (D3 WT) at day 3 after UUO. i A volcano plot indicated higher expression of extracellular matrix- and myofibroblast-associated genes (including Pdgfra , Col1a1 , Col1a2 , Col3a1 , Col4a1 , Col14a1 , Fn ) in WT mice than FibEGFR−/− mice at day 3 after UUO. Data are means ± SEM, ** P < 0.01, *** P < 0.001, analyzed using two tailed Student’s t test for ( c ); 2-way ANOVA followed by Bonferroni’s post hoc test for ( d ).

Article Snippet: All animal experiments were performed in accordance with the guidelines and with the approval of the Institutional Animal Care and Use Committee of Vanderbilt University. iRhom2−/− mice (C57BL/6J) were purchased from The Jackson Laboratory (#044040).EGFR floxed ( EGFR f/f ) mice were generated by flanking exon 3 of the EGFR gene with two LoxP sites, as described previously . iRhom2 knockout mice (strain#044040, C56BL/6), PDGFRβ-P2A-CreER T2 mice (Strain #: 030201, C56BL/6), and R26 LSL H2B mCherry (Rosa26-CAG-LSL-H2B-mCherry mice (Strain #023139) were all purchased from The Jackson Laboratory.

Techniques: Isolation, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Epidermal growth factor receptor activation is essential for kidney fibrosis development

doi: 10.1038/s41467-023-43226-x

Figure Lengend Snippet: a – d snRNAseq analysis indicated characteristics of fibroblast and myofibroblast subclusters following unilateral ureteral obstruction (UUO). The R package CellChat was used, and CellChatDB is the built-in database in CellChat without a separate version. a Gene Ontology (GO) Biological Process analysis for fibroblast subclusters based on the top marker genes for each cluster suggested that Fib1 cluster was associated with kidney development, MF3 cluster with immune cell activation and differentiation, and MF4 cluster with fibroblast proliferation. b Trajectory analysis showed a continuous change in RNA profile in WT fibroblasts from day 1 to day 3 after UUO injury. c Pseudotime analysis was performed to infer paths of cell-state transitions within the fibroblast clusters. d In combination with GO terms, Fib2/MF2 dominated the first day in the UUO model, probably due to the activation and differentiation of local fibroblast cells in the kidney. Fib1/MF3 may arise from the transdifferentiation of renal cells after injury or some cells from migration, and these fibroblast subclusters were more inclined to activate immune cells and promote differentiation and to participate in cross-talk with immune cells. MF4 was associated with fibroblast proliferation on tday three in the UUO model. e FibEGFR−/− had less kidney fibrosis compared to WT mice at day 3 after UUO. n = 4. Scale bar = 50 μm. Both the percentage of mCherry+ fibroblasts/myofibroblasts in total kidney cells ( n = 7) ( f ) and the proliferation rate of fibroblasts/myofibroblasts (EdU+ mCherry+ double positive cells) ( n = 4) ( g ) were markedly lower in FibEGFR−/− mice than WT mice. h Immunofluorescent staining showed fewer kidney PCNA+ α-SMA+ double positive cells (red arrows, proliferating fibroblasts/myofibroblasts) in FibEGFR−/− mice compared to WT mice. n = 5. Of note, PCNA+ positive cells were also observed in tubular cells in both WT and FibEGFR−/− mice. Scale bar = 50 μm. Data are means ± SEM, ** P < 0.01, ** P < 0.001, analyzed using two tailed Student’s t test for all.

Article Snippet: All animal experiments were performed in accordance with the guidelines and with the approval of the Institutional Animal Care and Use Committee of Vanderbilt University. iRhom2−/− mice (C57BL/6J) were purchased from The Jackson Laboratory (#044040).EGFR floxed ( EGFR f/f ) mice were generated by flanking exon 3 of the EGFR gene with two LoxP sites, as described previously . iRhom2 knockout mice (strain#044040, C56BL/6), PDGFRβ-P2A-CreER T2 mice (Strain #: 030201, C56BL/6), and R26 LSL H2B mCherry (Rosa26-CAG-LSL-H2B-mCherry mice (Strain #023139) were all purchased from The Jackson Laboratory.

Techniques: Marker, Activation Assay, Migration, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Wound infiltrating adipocytes are not myofibroblasts

doi: 10.1038/s41467-023-38591-6

Figure Lengend Snippet: 3D whole mount time-lapse imaging snapshots of single-cell tracks skin explants generated from Adipoq cre or En1 Cre crossed to R26 LSL-H2B-mCherry reporter mice. a Snapshots of adipocyte- and fibroblast-migration tracks on day 1. b Adipocyte and fibroblast tracks on day 4, generated by automated cell tracking using Imaris version 9.2. (Bitplane). c Manual tracks of adipocytes and fibroblasts in the scar region of explants at day 1 and day 4; the plot shows the difference in migration distance and type of movement in the scar region of both adipocytes and fibroblasts. N = 2 videos per time point. Scar regions were cropped (700 µm X 700 µm) from whole explant and cells manually tracked. Blue indicates starting time and red is the end-point. d 3 main types of movement quantified using manually annotated single cell tracks present in c, n = 3 biological repeats, mean ± SD. e Velocity of migratory adipocytes and fibroblasts is calculated using time-lapse videos and automated single cell tracks. Velocity variation and amplitude difference from time point 4 −9 hours across all samples are shown in higher magnification (lower panel). The red crosses (+) indicate the mean velocities of the indicated time points. f Spline graph of day 4 showing differences of mean velocity between adipocytes and fibroblasts. g Neighbour similarity analysis of day 4 explants using automated single-cell tracks generated from 3D time lapse videos. The colour bar represents the movement angles 0° (red, coordinated movement) to 90° (blue, random movement). Fibroblast migrations are coordinated and collective, whereas adipocyte migrations are random and individual. h Directed and non-directed movement of fibroblasts and adipocytes respectively at day 4. Scale bars:100 µm.

Article Snippet: En1Cre (JAX stock No. 007916), ROSA26LSL-H2B-mCherry (R26 LSL-H2BmCherry ) (JAX stock No. 023139), Rag2 –/– mice and ROSA26mTmG (R26 mTmG ) reporter mice were from Stanford University .

Techniques: Imaging, Generated, Migration, Cell Tracking Assay