Study On The Immortalization Of The Primarily Isolated Islets From SD Rats With SV40T Gene Transduction

Objective Our object is to establish an immortalized islet cell line with SV40T gene transduction.Methods SD rat islets were isolated using defined protocols for enzymatic dissociation with Collagenase V and purification using discontinuous Ficoll gradients density centrifugation.Islets viability and purification were performed with AO/PI and DTZ staining, respectively. The function of the islets was conducted with a glucose-induced insulin-releasing test. After transduction of the islets with SV40T gene for three days and selection with hygromycin for one week, we harvest hygromycin-positve cells then detect the expression of the SV40T and pancreatic specific transcriptional factors at mRNA and protein level through RT-PCR and Western-blot, respectively.Results Trough SV40T gene transduction,we obtained hygromycin-positve islets and passage them for 30 generations and the function of the islets were equivalent to the freshly isolated rat islets.Conclusion We successfully established a immortalized islets cell line with SV40T gene transduction.