Olgun Yağ Hücrelerinin Rekombinant Protein Ifade Kapasitelerinin İncelenmesi
Özet
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.
