IMMORTALIZATION OF YANGTZE FINLESS PORPOISE FIBROBLAST CELL AND PRELIMINARY STUDY ON THE GROWTH CHARACTERISTICS

The Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) is the sole freshwater subspecies of N. phocaenoides. As a mammalian species at the top of the food chain, it has been described as sentinels for the health of the Yangtze River ecosystem. Obtaining stable Yangtze River cell lines from fresh tissue is technically and legally difficult. In this study, we successfully established a finite cell line which deprived from placenta umbilical vein of the Yangtze finless porpoise, and an infinite cell line through transfection (simian virus 40 T antigens) techniques. The morphology, transfection efficiency, dynamic growth, and survival rate of the infinite cultured cells were extensively studied. The present study shows that prior direct establishment from fresh tissue leads to more efficient cell line establishment compared with enzymatic dissociation. Primary culture cells were isolated from the edge of the tissue for almost 14 days, cultured at 37℃ and 5% CO₂ in the standard mammalian medium DMEM supplemented with 10% fetal bovine serum (FBS), and monolayers were observed in 20 days. Cells were verified as fibroblasts based on their spindle-shaped morphology. After a limited number (less than 14) of transmissions, the primary fibroblast proliferation ceases to occur. Following the transfection of Yangtze finless porpoise primary fibroblasts with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. The cell line can propagat 40-50 times. We used PCR to amply the expression of SV40 Large T antigens. Population doubling time was about 24h and cells were successfully cryopreserved and thawed. Furthermore, we identified no significant difference in cell viability between different generations of immortalized fibroblasts before and after resuscitation with trypan blue staining. By transfecting the exogenous plasmid pEGFP-C1 and pERed2-N1, the efficiency of transfection of the foreign plasmid was about 15%, indicating that the exogenous gene can be expressed. The cell line of Yangtze finless porpoise is hard to be transfected using plasmid. In conclusion, we established a stable Yangtze finless porpoise cell line for the first time without hurting any Yangtze finless porpoise, and this cell line shares important features of the immortalized cell lines. Future research will focus not only on toxicity aspects but also on the development of immortalized Yangtze finless porpoise cell lines to enable long-term maintenance and application. Importantly, we provided a protocol for establishing a stable cell line for Yangzte finless porpoise so that we can explore the function of genes and molecular mechanisms related to Yangtze finless porpoise. The establishment of this stable cell line allows a wealth of functional studies on the unique mechanism of Yangtze finless porpoises, which can make it possible to optimizes advanced technology applications on the field of wild aquatic animals in protection.