The Research Of Regulatory Effects Of Porcine Insulin Promoter On Gene Expression In The Porcine Non-βcells

Human islet amyloid polypeptide (hIAPP), insulin (INS) gene were expressed only in β cells and their promoters could interact with some transcription factors which expressed in β cells. In the enhancer region of pig insulin gene promoter (PIP), there were many cis-acting elements which could interact with three transcription factors Pdx-1, MafA and NeuroD1. This experiment was to study the effect of three transcription factors, Pdx-1、MafA and NeuroDl on the regulation of pig insulin promoter (PIP) in non-β cells, PK15cell line and to study the expression level of exogenous gene hIAPP in PK15cells. This study could provide some useful data and reference for the research of hIAPP transgenic pigs and the research of other tissue-specific expression vectors.1) Analysis on the expression level of Pdx-1, MafA and NeuroDl in tissues of pigs. Results of Fluorescence quantitative PCR showed that gene expression level of Pdx-1, MafA and NeuroDl in pancreas were100,300and400times the level in muscle, lives and kidneys, respectively.2) Construct over-expression vectors of Pdx-1, MafA and NeuroDl to research their regulation on PIP. In vitro, over-expression vectors of Pdx-1, MafA and NeuroDl were constructed and transfected into PK15cells in different groups. Dual luciferase activity was detected and these three transcription factors could activate PIP in different extent. MafA and NeuroD1could activate PIP with synergistic effect while three of them could not activate PIP synergisticly.3) Detection of expression of hIAPP in mRNA and protein level. On the basis of dual luciferase activity, pGL3-Pdx-1、pGL3-MafA and pGL3-NeuroDl and pcDNA3.1-PIP-hIAPP were transfected into PK15cells in different groups to detect expression level of hIAPP. Results showed that hIAPP expressed in high mRNA level, while it can’t express mature hIAPP protein when detected by Western blot and RIA. This may because it doesn’t contain some key enzymes that involved in translation process of hIAPP in PK15cells.4) Detection of expression of endogenous INS in wild Wuzhishan pigs. Results of Fluorescence quantitative PCR showed that gene expression level of INS in pancreas were840、70and145times the level in muscle, lives and kidneys, respectively.This experiment indicated that Pdx-1, MafA and NeuroD1could activate PIP in PK15cells. hIAPP could not express mature protein in PK15cells may because PK15cells doesn’t contain some key enzymes that involved in translation process of hIAPP in PK15cells and it could provide some useful reference for the research of research of hIAPP transgenic pigs and the research of other tissue-specific expression vectors. At the same time, results of endogenous INS in wild Wuzhishan pigs were expressed in pancreas in tissue-specific way indicated that Pdx-1, MafA and NeuroDl could really activate PIP in PK15cells.