Journal: Biotechnology Reports

Article Title: Comparative analysis of anti-MICA scFv affinities: Insights from three label-free biophysical methods and biological validation

doi: 10.1016/j.btre.2026.e00955

Figure Lengend Snippet: Characterization of Recombinant Proteins: MICA and anti-MICA scFvs. (A) Molecular model, shown as a ribbon representation, of the variable fragment of the anti-MICA scFvs. The framework is displayed in white, the light chain CDRs are shown in cyan, and the heavy chain CDRs are shown in yellow. The residues with mutations are shown as magenta spheres [residues 32 (CDR L1), 164 (CDR H1), and 188/190 (CDR H2)]. (B) Schematic diagram of the scFv gene. The modified pET-15b vector was used for the expression of the WT and Beta mutant scFvs, each carrying four mutations: I32Y in CDR1 of the VL, and S164F, P188W, and G190W in CDR1, CDR2, and CDR2 of the VH, respectively. Recombinant proteins were expressed in E. coli BL21(DE3). (C) SDS-PAGE analysis showing the purity of recombinant proteins: WT scFv, Beta mutant scFv, and MICA. Proteins were resolved on a 12% acrylamide gel under reducing conditions. SDS-PAGE results show the soluble fraction (SF), unbound protein (UBP), elution of purified scFv (E), renatured proteins (R) and inclusion bodies (IB). MW, molecular weight. (D-E) Western blot analysis confirming the identity of scFvs and MICA using an anti-HisTag antibody. For the identification of the WT and Beta mutant scFvs, Anti-6xHis Epitope Tag mouse monoclonal antibody conjugated with peroxidase (200-303-382) was used at a dilution of 1:1000. For the identification of MICA, a biotinylated Anti-MICA antibody (BAMO3 (BAFI300, BamOmaB)) and Streptavidin were used at a dilution of 1:2000. A total of 2 μg of purified protein was loaded. The negative control (Ctrl -) for MICA detection was WT scFv and MICA protein was used for scFv detection. Original gel is presented in Fig. S1, Supplementary information.

Article Snippet: The identity of MICA and scFvs proteins was confirmed by western blot using a HRP-conjugated anti-His tag monoclonal antibody (200-303-382, Rockland, USA).

Techniques: Recombinant, Modification, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Acrylamide Gel Assay, Purification, Molecular Weight, Western Blot, Negative Control

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