The Establishment of Asian Black Bear (Ursus thibetanus) Fibroblast Cell Line

The Asian black bear (<i>Ursus thibetanus</i>, UT), a Class II protected species in China, presents significant challenges for dermatological research due to limited availability of skin specimens and technical difficulties in fibroblast isolation. To address this, this study developed an immortalized fibroblast cell line through lentiviral-mediated introduction of human telomerase reverse transcriptase (hTERT). The hTERT gene fragment was amplified via PCR and cloned into a eukaryotic expression plasmid using homologous recombination. The recombinant plasmid was packaged into lentiviral particles using a three-plasmid system (psPAX2, pMD2.G) for stable transduction of primary <i>Ursus thibetanus</i> skin fibroblasts (UTSF). Puromycin selection enabled isolation of positive clones, which were subsequently expanded to establish an immortalized cell line. Comprehensive validation through Western blotting, PCR and immunofluorescence confirmed successful genomic integration and sustained expression of hTERT. This study successfully immortalized the skin fibroblasts of black bears and established a fibroblast cell line of Asian black bears. This immortalized UTSF cell line provides a critical in vitro model system for investigating cutaneous physiology and pathogen interactions in this protected species.