Construction of a high efficient pancreatic duodenal homeobox 1/insulin dual-reporter vector and its preliminary application / 中国组织工程研究

ABSTRACT

BACKGROUND:Islet beta cell replacement therapy is one of the most promising approaches for treating type 1 diabetes mellitus. However, its large scale application is hampered by a shortage of islet beta cells for transplantation. Pluripotent stem cells are one of ideal seed cells for islet beta cell replacement therapy, but pancreatic beta-cell differentiation is time-consuming and labor-intensive. OBJECTIVE:To construct a high efficient pancreatic and duodenal homeobox 1 (Pdx1)/insulin dual-reporter vector and to monitor the key genes expression during pancreatic beta-cell differentiation from pluripotent stem cells. METHODS:In order to construct a high efficient Pdx1/insulin dual-reporter vector, puromycin resistance gene was firstly introduced into pTiger vector, and then the original 410 bp mouse Ins1 promoter of the vector was replaced by 646 bp mouse Ins1 promoter. Finally, the dual-reporter vector was transduced into INS-1 and human induced pluripotent stem cells to testify its function. RESULTS AND CONCLUSION:The high efficient Pdx1/insulin dual-reporter vector was constructed successfully. The vector successfully acquired puromycin resistance gene and high gene expression efficacy of insulin in INS-1 cells. The specific gene expression pattern of Pdx1/insulin was first found in INS-1 cells. To conclude, the real-time monitoring function of Pdx1/insulin expression is preliminarily confirmed during pancreatic beta-cell differentiation.