POLISTRONIC DESIGN, IN VITRO SYNTHESIS AND CHARACTERIZATION OF SYNTHETIC mRNAs OF PANCREATIC BETA CELL GENES


Thesis Type: Postgraduate

Institution Of The Thesis: Akdeniz University, Faculty of Agriculture, Department of Agricultural Biotechnology, Turkey

Approval Date: 2021

Thesis Language: Turkish

Student: Fatma AKÇAKALE

Principal Supervisor (For Co-Supervisor Theses): Mehmet Fatih Cengiz

Abstract:

Hücrelere DNA veya RNA’yı etkili ve güvenilir bir şekilde in vitro ve in vivo koşullarda aktarmaEffective and reliable methods are required to transfer genetic materials such as  DNA and RNA into to cells. Gene transfer methods are divided into two groups: viral and non-viral methods. In viral methods, the natural contagion of viruses is used to transfer genetic material to cells. The biggest limitations of viral methods are their potential immunogenicity and random insertion into the genome. Non-viral methods includes the use of physical or chemical compounds to transfer genetic material into the target cells. Chemical methods are generally less immunogenic and relatively safer than viral vectors. mRNA generated by in vitro transcription in non-viral methods is an effective method for using plasmid DNA instead in therapeutic gene transmission, as it can be easily obtained with commercially available kits. By combining various mRNA modifications and transfer methods, the effectiveness of mRNA gene therapy has been greatly improved. For beta-cell differentiation, three major transcription factors (PDX1, NGN3 and MAFA) has to be expressed within the cell. In previous studies where these transcription factors were transfected monocıstronically, reprogramming resulted in low efficiency. In this study, it was aimed to increase the reprogramming efficiency by using polycistronic mRNA technology. The aim of this thesis study is to design mRNAs of PDX1, NGN3 and MAFA transcription factors as polycistronic, in vitro synthesis and characterization by AR42J-B13 cells. As a result of the studies, synthetic pmRNA design and in vitro synthesis were successfully performed and the activation of endogenic Pdx1, Ngn3 and MafA genes were successfully demonstrated.