Journal: bioRxiv

Article Title: Unbiased Long-Read Whole-Genome Sequencing Enables High-Resolution Mapping of Transgene Concatenation and Off-target Genomic Disruption in a Mouse Model

doi: 10.64898/2026.05.15.725597

Figure Lengend Snippet: (A) Schematic of pCAG-ZZEF1-ALOX15-T2A-L2T designed to express the ZZEF1-ALOX15 fusion gene, luciferase and tdTomato under the control of the CAG promoter. (B) In vitro validation of the construct. Co-transfection of HEK293T cells with pCMV-Cre activated tdTomato reporter expression, confirming construct functionality. Images were captured at 10x original magnification; scale bars, 750 μm. (C) Gel electrophoresis and Sanger sequencing alignment (Geneious Prime) following PCR genotyping of retinal DNA from an HRGP-Cre+/-;ZZEF1-ALOX15+/- mouse, showing excision of the loxP-flanked stop cassette. Raw gel electrophoresis results are provided in Supplementary Figure S5. (D) Top panel: Fundus and OCT examination of both eyes of an HRGP-Cre+/-;ZZEF1-ALOX15-/- littermate control at week 54. The small yellow spot in the right eye fundus is suggestive of drusenoid lesion development. Bottom panel: Both eyes of an HRGP-Cre+/-;ZZEF1-ALOX15+/- mouse showing dots on fundus corresponding to hyper-reflective drusenoid lesions (*) at the RPE. Retinal lamination appears preserved across both transgenic and control mice. (E) Fundus imaging of both eyes at week 5 showing a littermate control (Dkk3-Cre+/-;ZZEF1-ALOX15-/-) in the top panel and a transgenic mouse ( Dkk3-Cre+/-;ZZEF1-ALOX15+/- ) in the bottom panel. Both eyes appear unremarkable on examination. Imaging with a tdTomato excitation filter over the same region reveals no detectable fluorescence. (F) Flat-mount cryo-immunohistochemistry (cryo-IHC) showing absence of tdTomato reporter in both transgenic lines. In HRGP-Cre+/-;ZZEF1–ALOX15+/- sections (8 weeks), nuclei were stained with DAPI (cyan), rods with rhodopsin (purple), cones with cone arrestin (green), and tdTomato (red). In Dkk3-Cre+/-;ZZEF1–ALOX15+/- sections (11 days), nuclei were stained with DAPI (cyan), rods with rhodopsin (green), cones with cone arrestin (white), and tdTomato (red). Images were captured at 40x original magnification; scale bars, 20 μm.

Article Snippet: For ex vivo evaluation, the retina and retinal pigment epithelium (RPE) tissue was collected at P11 from a control ( Dkk3-Cre +/- ;ZZEF1-ALOX15 -/- ) and transgenic littermate ( Dkk3-Cre +/- ;ZZEF1-ALOX15 +/- ) for immunohistochemistry (IHC) to assess reporter expression and cellular morphology as previously described except that retinal cryosections were incubated with tdTomato antibodies (Origene; AB8181-200, 1:400) instead of GFP in combination with Cone Arrestin (Millipore-Sigma; AB15282; 1:1000), and Rhodopsin (Sigma-Aldrich; O4886, 1:10,000) .

Techniques: Luciferase, Control, In Vitro, Biomarker Discovery, Construct, Cotransfection, Expressing, Nucleic Acid Electrophoresis, Sequencing, Transgenic Assay, Imaging, Fluorescence, Immunohistochemistry, Staining

Journal: Cell reports

Article Title: Stc1-expressing myofibroblasts are a developmentally distinct lineage cleared through apoptosis in the neonatal lung

doi: 10.1016/j.celrep.2025.116750

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) of lung mesenchyme at 12 developmental time points from 3 merged scRNA-seq datasets. The dashed line surrounds myofibroblast cell types. (B) Gene expression of Stc1 in myofibroblast cell types from analysis in (A) at P3, P5, and P7. (C) Diagram of Stc1 locus targeted to generate a knockin Stc1 CreERT2 allele. (D) Experiment schematic for (E)–(I). (E) Stc1-lineage-labeled lung harvested at P7 and immunostained for SM22. The arrows indicate Stc1-lineage-labeled cells that are also positive for Pdgfra GFP and SM22. Z-projections of approximately 100 μm (top) and 40 μm (bottom) are shown. (F and G) Flow cytometry analysis (F) and quantification (G) of all live cells in the P7 lung for Stc1-lineage tdTomato cells positive for Pdgfra GFP (“SCMF”) or negative for Pdgfra GFP (“other”). n = 3, mean ± SD. (H and I) Flow cytometry analysis (H) and quantification (I) of mesenchymal cells in the P7 lung for Stc1-lineage tdTomato and Pdgfra GFP (“SCMF”). n = 3, mean ± SD. Stc1-lin, Stc1 lineage; aw, airway; bv, blood vessel; SCMF, secondary crest myofibroblast; DMF, ductal myofibroblast; ASM, airway smooth muscle; VSM, vascular smooth muscle; adv., adventitial; alv., alveolar; AOH, arm of homology. Scale bars: (E, top row) 50 μm and (E, bottom row) 10 μm.

Article Snippet: tdTomato Goat Polyclonal Antibody , Origene , Cat# AB8181-200; RRID: AB_3206272.

Techniques: Gene Expression, Knock-In, Labeling, Flow Cytometry

Journal: Cell reports

Article Title: Stc1-expressing myofibroblasts are a developmentally distinct lineage cleared through apoptosis in the neonatal lung

doi: 10.1016/j.celrep.2025.116750

Figure Lengend Snippet: (A) Diagram of Stc1 CreERT2 -mediated conditional knockout of a floxed Bax allele, in a background of Bak1 knockout, Pdgfra GFP , and Rosa tdTomato . (B and C) Stc1-BB Het (B and C) and Stc1-BB KO (B′ and C′) left lung cross-sections at P16 (B and B′) and P30 (C and C′). Z-projections of approximately 100 μm are shown. (D and E) Higher-magnification confocal microscopy of Stc1-BB Het (D and E) and Stc1-BB KO (D′ and E′) left lung cross-sections at P16 (D and D′) and P30 (E and E′). Z-projections of approximately 30 μm are shown. (F) Quantification of Stc1 lineage in Stc1-BB Het and Stc1-BB KO lungs. Blood vessels were excluded from analysis. n = 4–5 (P16) and 7–8 (P30), mean ± SD. (G) H&E staining of Stc1-BB Het and Stc1-BB KO lungs at P30. (H) Quantification of alveolar wall thickness from H&E staining in Stc1-BB Het and Stc1-BB KO lungs at P30. n = 4–6, mean ± SD. Stc1-lin, Stc1 lineage; Tam, tamoxifen; Het, heterozygous; KO, knockout; aw, airway; bv, blood vessel; ns, not significant. Scale bars: (B–C′) 500 μm, (D–E′) 50 μm, and (G) 100 μm. Statistics: ns p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: tdTomato Goat Polyclonal Antibody , Origene , Cat# AB8181-200; RRID: AB_3206272.

Techniques: Knock-Out, Confocal Microscopy, Staining