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• W2089543125 abstract "36 A novel approach to gene therapy for insulin dependent diabetes mellitus (IDDM) is to transplant genetically modified human liver cells in the patients. This therapy requires the development of human liver cells to replace beta cells that can synthesize and process active insulin (mature insulin). Human liver cells cannot simply process proinsulin cDNA to make mature insulin because they lack the endoprotease to cleave the B-chain-C-peptide junction and C-peptide-A-chain junction. To achieve this link we used the human hepatoma cell line (HepG2) as a model to genetically engineer the human insulin gene. Methods: PCR-based site-directed mutagenesis was performed to create two mutation sites on insulin cDNA between the B-chain and C-peptide and between the C-peptide and A-chain, which were recognized by furin endoprotease in HepG2 cells. The mutated and wild-type insulin cDNA were cloned into mammalian expression vectors driven by the CMV promoter, respectively. In the permanent transfection of HepG2 with the mutated insulin cDNA construct, the mature insulin was secreted into the media and radioimmunoassay (RIA), (Linco Research Inc, St. Louis MO), was used to detect the mature insulin. Results: The concentration of mature insulin that accumulated in the culture media increased and reached a peak on day 5 at 38uU/ml without changing media (3.5 fold above background). No active insulin was detected in transfected cells with wild-type cDNA and untransfected cells. Production of mature insulin was confirmed by 35S-Cysteine labeling of these transfected cells in vivo, followed by immuno-precipitation, SDS-PAGE, and phosphor imaging. A4-Kd mature insulin band was observed from the media of HepG2 cells transfected with mutated insulin cDNA. However, in the media of HepG2 cells transfected with wild-type insulin cDNA only a 9-Kd proinsulin band was found. This is evidence that HepG2 cells process mutated insulin cDNA to be mature insulin efficiently. Application of conditioned media containing mature insulin to stimulate normal HepG2 cells, which express the insulin receptors, confers a biological response. The beta unit (cytosolic domain) of insulin receptor was immuno-precipated and subsequently subjected to Western blot to examine phosphotyrosine protein. It was shown that there was a specific 100-Kd band comparable to control cells treated with media alone. Conclusions: We demonstrated that mutated human insulin cDNA can be produced in human HepG2 cells correctly and efficiently. This genetically modified insulin is biologically functional. These techniques will be applied to cultured normal human hepatocytes with a replication stimulus, in future experiments." @default.
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• W2089543125 date "1999-05-01" @default.
• W2089543125 modified "2023-10-14" @default.
• W2089543125 title "SYNTHESIS AND PROCESSING OF GENETICALLY MODIFIED HUMAN INSULIN BY HUMAN HEPATOMA CELL LINES (HEPG2)" @default.
• W2089543125 doi "https://doi.org/10.1097/00007890-199905150-00061" @default.
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