| Budget Amount *help |
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2005: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2004: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 2003: ¥5,900,000 (Direct Cost: ¥5,900,000)
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| Research Abstract |
We tried to establish an efficient method for inducing differentiation of embryonic stem (ES) cells into insulin-producing cells. As a tool of investigation, we established an ES cell line by introducing the expression unit, in which exogenous pdx-1 expression was precisely regulable by the Tet-off system, into the ROSA26 locus.
Using this cell line, we examined the effect of pdx-1 expression during in vitro differentiation via embryoid body formation. The results showed that pdx-1 expression clearly enhanced the gene expression of the insulin 2, somatostatin, Kir6.2, glucokinase, neurogenin3, p48, Pax6, PC2, and HNF6 genes in the differentiated cells. However gene expression level of insulin gradually decreased as cell culture passaged. To solve this problem, we constructed adenoviral (AdV) vectors expressing several transcription factors relating the differentiation of pancreatic β cells. Induction of neuroD gene to this cell line by AdV vector showed up-regulation of gene expression of insulin 2, insulin 1, glucagon and somatostatin.
To improve the efficiency of differentiation, we established ES cell line in which exogenous sox17 expression was regulated by tetracycline. Induction of pdx-1 and mafA genes by adenoviral vectors to sox17-expressing ES cells induced the expression of insulin 2 gene.
Thus, drug-regulable system of gene expression in these ES cell lines may be a useful tool for investigating the genes for early development and differentiation of pancreatic β cells.
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