Olgun Yağ Hücrelerinin Rekombinant Protein Ifade Kapasitelerinin İncelenmesi

Abstract

This study postulates that the extracellular transport of the secretory proteins of the adipocytes (adipokines) can be utilized to achieve recombinant protein expression. Adipokines are exported outside of the adipocytes by the help of leader sequences which deduce the extracellular export traffic. It is aimed to achieve the expression of a recombinant protein extracellularly by using the leader sequences of the 7 selected adipokines. This approach will aid in production and extracellular secretion of the protein of interest by mature adipocytes. The efficiency and intracellular- extracellular follow-up of these export signals will be done by fusing these export signals to N- terminal of Green Fluorescence Protein (GFP). The S/MAR sequences increase the episomal persistence of the transgenic vector where it is located to and prolong its expression in mammalian cells. In this study, it is aimed to use this property of S/MAR sequences by adding it to the vector construct to increase the episomal expression of the transgenes in mature adipocytes (non-dividing cells). It is proposed to compare the efficacy of two different S/MARs in this study, the S/MAR sequences of Apolipoprotein B (APO-B) and Interferon B (IFN-B) genes. The efficiency of this designed recombinant gene expression construct will be shown by expressing the dominant-negative form of myostatin (MSTNdn), as a model of gene of interest, in adipocytes and observing its effect on myoblast cells. As a result of this study, export signal of Retinol Binding Protein (RBP4) has been selected as the most efficient export signal. No differences have been observed between the selected S/MAR sequences. At the end of this study, the in vitro efficiency of the designed vector construct containing selected export signal (RBP4), S/MAR sequences, and MSTNdn was shown by implementing the secretion of this model recombinant protein from the transfected mature adipocytes.