Establishment And Evaluation Of Immortalized Dzo Kidney Cell Line

The interspecific F1 generation hybrid of Bos Cattle-yak as a cross between yak and yellow cattle exhibited distinct characteristics in terms of physical appearance,fitness,production performance,disease resistance,growth and development,and reproduction rate,as well as being able to adapt to a harsh weather environment.As a species endemic to Northwest China,its value has been underutilized.However,as dzo kidney cells with culturable BVDV virus,their ability to divide under in vitro culture conditions is limited,and they stop proliferating into senescence after a certain period of growth.Senescence is a major barrier to the immortalization of primary cells unable to proliferate in vitro,while telomerase prevents telomere erosion,preventing telomere controlled senescence.At present,exogenous h TERT can activate telomerase activity to allow cells to bypass the senescence phase without causing significant changes in phenotypic properties.In addition to this,the LT antigen of SV40 is also one of the ways in which cells can be immortalized,not only by binding to the RB-E2 F complex to activate E2 F mediated transcriptional activation,but also by blocking these two mechanisms of transcriptional activation of p53 to allow cells to overcome growth arrest,prevent apoptosis and lead to cell proliferation.Therefore,to further develop the utilizable value of Bos Cattle-yak,taking into account that the cellular matrices(e.g.,MDCK cells and VERO cells)currently applied for vaccine production are all kidney cells,in this study,the construction and evaluation of immortalized cell lines were performed using three methods: spontaneous passage,liposomal transfection,and lentiviral transfection.The main results were obtained as follows:1.The optimal culture conditions for the dzo kidney cells were selected to be a passage ratio of 1:3 and 10% fetal bovine serum by culturing the cells through three variables including different passage ratios,different serum and different serum concentrations.Following the screened optimal culture conditions,spontaneous immortalization of the dzo kidney cells was attempted,but unsuccessful,and eventually passaged to passage F17.Cell cycle and apoptotic cell identification of the dzo kidney cells at different passages revealed that the dzo kidney cells gradually decreased their proliferative activity and finally became apoptotic during spontaneous passaging.2.Using a liposomal transfection method,the plasmid pcl-neo-CMV-EGFP-Linker-h TERT is transferred into dzo kidney cells at the F3 generation,but liposomal transfection modalities were unsuccessful,collectively termed NZLT cells.Using the lentiviral transfection method,attempts were made to transfer h TERT into dzo kidney cells,which were successfully immortalized by puromycin selection,but the cells were not immortalized and only passaged to the F10 passage,collectively NBLT cells.SV40-LT was attempted to be transduced into dzo kidney cells by lentiviral transfection method,after puromycin selection,the immortalization way of lentiviral transfection SV40-LT was successful,it has been serially passaged up to 50 passages,collectively NBLS cells.3.Five aspects of morphology,gene stability,growth characteristics,genetic characteristics and functional validation were analyzed: NBLT cells did not show altered morphology,h TERT gene levels and protein levels were high,genetic characteristics remained stable,but cell proliferation rate weakened.NBLS cells have an elongated morphology,high SV40-LT gene and protein levels during passaging,stable genetic characteristics,accelerated cell growth and proliferation,and increased viral infection efficiency.In summary,in this assay,h TERT with the SV40-LT gene was successfully introduced into the cell genome,but only SV40-LT induced activation of telomerase in cells,prevented telomere shortening and maintained a sufficient and stable length,and finally allowed the cells to immortalize,resulting in the successful construction of dzo kidney immortalized cell lines and laying the foundation for subsequent studies.