The Action of Cytomegalovirus (CMV) Promoter on Expression of Genetically Engineered Insulin in Rat Hepatocytes
Keywords:Cloning, CMV promoter, Gene therapy, Hepatocytes, Mutation, Non-viral Vector, Preproinsulin, Transfection
Background: Insulin is a hormone produce in the pancreas that stimulates glucose uptake from blood to enter the body's cells, where it is converted into energy needed by muscles and tissues to function. The pancreatic cells are responsible for the secretion of insulin. In diabetes, the body cannot produce enough insulin or cannot use insulin effectively.
Objectives: In this study, we attempted to activate the hepatic cells to secrete insulin instead of the pancreatic cells.
Methodology: This was done by gene therapy; an excellent strategy to treat diabetes by supplying the correct wild type copy of a furin cleavable sites preproinsulin. The preproinsulin was extracted from the rat DNA, cloned and mutated to generate the two furin cleavable sites responsible for removal of C-peptide to form the two chains (A and B) for mature insulin production. This mutated insulin was derived by Cytomegalovirus (CMV) promoter to express insulin by its transfection inside the primary rat hepatocytes using a non-viral vehicle to keep the hepatic cells healthier against the transfection. In vitro, the rat hepatocytes could not divide well as in vivo, but with special hormones like insulin and dexamethasone lived longer and kept their function.
Results: The CMV promoter is strong and lead to over expression of mature insulin inside rat hepatocytes leading to deterioration, toxicity of hepatocytes and finally cell death. Hepatocytes in vitro were more fragile and needed some modification to adapt with secretion of insulin.
Conclusion: Glucokinase or glucose transporter promoters were much more perfect than CMV. They can activate the hepatocytes to modulate the glucose level and so limiting the amount of insulin secreted. These promoters are weaker than CMV but much more perfect for hepatocytes.