EVALUATION OF PX458 LIPOFECTION AND PUROMYCIN SELECTION IN BOVINE OVIDUCT EPITHELIAL CELLS - Qurat ul Ain; Afshan McCarthy; Asif Nadeem; Kathy Niakan; Wasim Shehzad; Tahir Yaqub; Ali Fouladi Nashta; Maryam Javed (Journal of Animal and Plant Sciences, 2026)
The epithelial cells play a significant role in the developmental and reproductive functions of the mammalian oviduct. Recent innovations in tissue culture methods have enabled long-term in vitro culturing of the bovine oviductal epithelial cells (BOECs). The sensitive phenotype and limited proliferative capacity of BOECs made efficient plasmid delivery challenging. To provide robust experimental platforms for gene-function and mechanistic studies, a reproducible, well-defined primary culture with optimised lipofection protocols has been established. In this study, we isolated primary BOECs from fresh cow oviducts, cultured and passaged them to optimise lipofection conditions and evaluate plasmid delivery and transfection efficiency. Lipofectamine™ Stem Transfection Reagent with pX458 and pMax-GFP plasmids was used to perform the transient transfection. We assessed a range of cell seeding densities (0.75-1.5×10⁵ cells/ml/well) and plasmid concentrations (1-2.5 µg per well) to evaluate lipofection efficiency. The cell viability was assessed using the Trypan Blue exclusion assay and morphological evaluation of cultured cells, and GFP expression was monitored qualitatively using fluorescence microscopy. Our findings showed that 2.0 µg of pMax and 1.5 µg of pX458 with 1 × 10⁵ cells per well exhibited greater GFP expression with minimal cytotoxicity. Lipofection of BOECs resulted in generally lower transfection efficiency, with pX458 exhibiting markedly lower performance than pMax. However, the successful detection of the GFP expression using pX458. Successful detection of GFP expression from pX458 supports its use as a suitable reporter model for related CRISPR/Cas9 vector systems used in genome-editing studies. A puromycin kill curve was performed to determine the effective antibiotic concentration for bovine oviductal epithelial cells, providing a basis for future CRISPR-based genetic manipulation studies.
Keywords: journal article; Bovine Oviductal Epithelial Cells (BOECs); Lipofection optimisation; pX458; pMAX-GFP