Designing an engineered gene construct sensitive to carbohydrate in vitro and candidate for ...

This study aim is to prepare a suitable high- performance vector for gene therapy in diabetes mellitus. The designed vector has prominent characteristics, such as directed replacement, which makes it a suitable method for treating or preventing other genetic disorders. Materials and Methods: Whole rDNA sequence of the human genome was scanned. The 800 bp two homology arms were digested by EcoRI and synthesized and cloned into the pGEM-B1 plasmid (prokaryotic moiety). The carbohydrate sensitive promoter, L-pyruvate kinase, and insulin gene were sub-cloned between homologous arms (eukaryotic moiety). The PGEM-B1 plasmid was digested by EcoRI, and the eukaryotic fragment was purified and transfected into Hela cell and cultured. The 300µg/ ml of glucose was added to the culture medium. Insulin expression in transfected cells with 200 and 400 ng of the construct, in compare with negative control was detected using western blot and ELISA. Results demonstrated that insulin was expressed toward glucose concentrates.
Source: Iranian Journal of Pharmaceutical Research - Category: Drugs & Pharmacology Source Type: research