Designing and in vitro production of important pancreatic transcription factors' mrnas and investigation of their roles in beta cell formation

Direct reprogramming of pancreatic beta cells has a great interest due to its possible use in diabetes treatment. Various studies demonstrated direct reprogramming of insulin expressing beta cells from other somatic cells by over-expressing important pancreatic transcription factors through viral vectors. However, concerns about using viral vectors in clinic have let the scientist to find new approaches for gene delivery and expression. In vitro synthesis and transfection of mRNAs is a safer strategy for ectopic gene expression. In this thesis study we pursued to design and in vitro synthesize human pancreatic transcription factors' mRNAs; Pdx1, Ngn3 and MafA. We also aimed to assess their expression efficiency in cultured cells. First we ligated the open reading frames of these genes to modified 5′ and 3′ UTRs. Ligation products were then cloned into a plasmid and subjected to in vitro transcription to get their corresponding mRNAs. Modified mRNAs were subsequently examined for their quality, quantity, and protein translation efficiency. In vitro-transcribed mRNAs were successfully transfected to the human fibroblast cells. Efficiently translated proteins were correctly localized into the nucleus. It is concluded that pancreatic mRNAs designed and in vitro-transcribed in this study can be used in further direct beta cell reprogramming studies.