Journal: Poultry Science

Article Title: LncRNA HAND2OT encoding micropeptide regulates the estradiol secretion, proliferation and apoptosis of ovarian follicular theca cells in chickens

doi: 10.1016/j.psj.2025.106206

Figure Lengend Snippet: The lncRNA HAND2OT encodes a micropeptide. (A) The predicted results of protein-coding ability of HAND2OT by CNCI and CPC2. (B) Schematic diagram of the ORF of HAND2OT predicted based on NCBI ORF Finder. Construction diagrams of the fusion vectors used to verify protein encoding (C, F, I). In pEGFP-MUT and ORF1/2/3+pEGFP-MUT vectors, the initiation codon (ATG) of EGFP and the termination codon (TAA/TGA) of HAND2OT ORF1/2/3 were deleted (C), and the ORF2-MUT vector was constructed based on the ORF2+pEGFP-MUT vector by mutating the start codon (ATG) of ORF2 to ATT (F). In ORF1/2/3-3 × Flag vectors, the termination codon (TAA/TGA) of HAND2OT ORF1/2/3 were deleted (I). Fluorescence imaging and western blotting demonstrating EGFP expression in chicken theca cells (D, E, G, H). The scale bars are 200 μm. (J) Western blotting demonstrating Flag protein expression in chicken theca cells. (K) Schematic illustration of the predicted amino acid sequence of the HAND2OT-66aa micropeptide encoded by ORF2.

Article Snippet: The PVDF membrane was incubated with anti-EGFP tag mouse mAb (Engibody, Shanghai, China) at 1:6000 dilution, anti-FLAG tag mouse mAb (Engibody, Shanghai, China) at 1:4000 dilution, and mouse anti-GAPDH monoclonal antibody (Proteintech, Wuhan, China) at 1:50,000 dilution.

Techniques: Plasmid Preparation, Construct, Fluorescence, Imaging, Western Blot, Expressing, Sequencing