Construction Of Eukaryotic Fluorescent Expressing Vector Of Simian Virus 40 Large T Antigen And Examination Of Rat Pancreatic Islet Cells Transfection

Objective To construct eukaryotic fluorescent expressing vector of simian virus 40 large T antigen gene(SV40T) and transfect rat pancreatic islet cells with this plasmids in order to detect changes of biological characteristic of pancreatic islet cells.Methods SV40T in plasmid of pCMV-SV40T was inserted into plasmid of pSC-A by digestion with Notâ… and Xhoâ… ,then SV40T in plasmid of pSC-SV40T was inserted into plasmid of eukaryotic fluorescent expressing vector pIRES2-ZsGreen1 by digestion with Xhoâ… and Sacâ…¡,further transfected into rat pancreatic islet cells with lipofectin.Expression of fluorescent and SV40T in transfectants was examined by fluorescene microscopy,RT-PCR and immunofluorescent.Changes of biological characteristic were examined by detection of basic insulin secretion and insulin secretion response to glucose stimulation,as well as immunofluorescent and clone formation test in soft agar.Gene changes examined by RT-PCR.Results After digestion with Xhoâ… and Sacâ…¡,the recombinant DNA was digested into two fragments(5.3kb,2.4kb),which matches the theoretical calculation.The life span of rat pancreatic islet cells transfected with the recombinant was extended.After being selected by G418,mRNA and protein of exogenous SV40T gene could be detected by RT-PCR and immunofluorescent.Decrease of insulin secretion was detected by ELISA.No clone was observed in soft agar.Trscription of Ngn3,which is specific for pancreatic islet progenitor cells, was detected by RT-PCR of later immortalized pancreatic islet cells.Conclusion The eukaryotic expressing vector of SV40T was constructed successfully and could be expressed in rat pancreatic islet cells.The life span of rat pancreatic islet cells transfected with the recombinant was extended,but functions degraded.Reversion to precursor cells of the immortalized pancreatic cells was detected.