Establishment of a non-invasive mouse reporter model for monitoring in vivo pdx-1 promoter activity.
It is well known that pancreatic and duodenal homeobox gene-1 (PDX-1) plays a crucial role in beta-cell differentiation, and maintaining mature beta-cell function. Thus, it is important to understand how pdx-1 gene is regulated under various pathophysiological conditions in vivo. In this study, to non-invasively and quantitatively monitor pdx-1 promoter activity in vivo, we constructed a pdx-1 promoter-SEAP-IRES-GFP reporter plasmid. In this construct, the -4.6kb pdx-1 promoter region sufficient for driving beta-cell-selective PDX-1 expression was inserted to the upstream of the secreted alkaline phosphatase (SEAP) reporter gene. It is noted here that the pdx-1 promoter-mediated SEAP activity can be distinguished from endogenous alkaline phosphatase activity. First, we transfected the construct in mouse beta-cell line MIN6 and human hepatocellular carcinoma cell line HepG2. SEAP activity was readily detected in the media of MIN6 cells, but not in HepG2 cells. These results indicate that this construct specifically reports beta-cell-specific pdx-1 promoter activity in a cell culture system. Based on these in vitro findings, we next generated transgenic mice using the same construct. SEAP activity was readily detected in serum of the transgenic mice, but not in their littermate mice. Furthermore, SEAP activity was detected in protein extract from the transgenic pancreas and slightly from the transgenic duodenum, but not from the liver, and brain. These results indicate that serum SEAP activity likely represents in vivo pdx-1 promoter activity. This transgenic mouse model would be useful to non-invasively monitor in vivo pdx-1 promoter activity and to screen new molecules which regulate PDX-1 expression.
Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Toshihiko
T
Shiraiwa
Hideaki
H
Kaneto
Takeshi
T
Miyatsuka
Ken
K
Kato
Kaoru
K
Yamamoto
Ayaha
A
Kawashima
Yoshitaka
Y
Kajimoto
Taka-Aki
TA
Matsuoka
Munehide
M
Matsuhisa
Yoshimitsu
Y
Yamasaki
Yoshio
Y
Fujitani
D000469:Alkaline Phosphatase / Q000097:blood / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D023421:Models, Animal; D008957:Models, Genetic; D010179:Pancreas / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D018398:Homeodomain Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase