title: Generation of fibroblasts overexpressing liver-specific PEPCK in a miniature             pig model of human type 2 diabetes mellitus.
abstract: Type 2 diabetes mellitus (T2DM) is one of the most common complex metabolic disorders in humans, and is characterized by hyperglycemia and metabolic alterations. In T2DM, fasting hyperglycemia is attributed to excessive hepatic glucose production, and increased gluconeogenesis has been ascribed to increased transcriptional expression of phosphoenolpyruvate carboxykinase (PEPCK). In this study, we analyzed porcine PEPCK promoter activities to generate liver-specific expression vectors. We generated miniature pig fibroblasts overexpressing PEPCK via transgenes to provide an animal model of human T2DM. Various regions of the promoter showed high levels of activity in the presence of glucocorticoids, a PEPCK gene inducer, in liver cells compared to a positive control promoter. The selected promoter region for a liver-specific expression system was adopted based on the current targeting vector containing two selection markers, green fluorescence protein and a neomycin-resistance gene. The linearized vector was introduced into pig fibroblasts which facilitated liver-specific PEPCK overexpression and screening according to the two selection markers. In the present study, we used a liposome-mediated transfection protocol rather than a virus-mediated gene delivery system, since the virus may cause side effects. Following transfection, 46 colonies out of 33 transfection trials had positively integrated the overexpression vector, indicating that a relatively high transfection efficiency rate was obtained by the liposomal-mediated system. Thus, we recommend the optimal liver-specific expression system for safe and effective transfection of pig cells. We plan to use these cells for somatic cell nuclear transfer to produce piglets that overexpress liver-specific PEPCK as an animal model for human T2DM.
authors.affiliation: SooAm Biotech Research Foundation, Seoul 137-851, Republic of                     Korea.
authors.forename: Yu-Kyung
authors.initials: YK
authors.lastname: Kim
authors.forename: Geun-Shik
authors.initials: GS
authors.lastname: Lee
authors.forename: Eui-Man
authors.initials: EM
authors.lastname: Jung
authors.forename: Sang-Hwan
authors.initials: SH
authors.lastname: Hyun
authors.forename: Woo-Suk
authors.initials: WS
authors.lastname: Hwang
authors.forename: Eui-Bae
authors.initials: EB
authors.lastname: Jeung
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D018390:Gene Targeting; D006801:Humans; D008099:Liver / Q000378:metabolism*; D009928:Organ Specificity; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013552:Swine; D014162:Transfection
chemical_list: D010729:Phosphoenolpyruvate Carboxykinase (GTP)