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A cell‐based assay system for high‐throughput screening of anti‐wrinkle agents in human dermal fibroblast transfectant cells
Author(s) -
Huh Sungran,
Lee Jongsung,
Jung Eunsun,
Ham Yeonggeun,
Kim Sang Suk,
Hyun Chang Gu,
Kim Yeong Shik,
Park Deokhoon
Publication year - 2007
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20060122
Subject(s) - luciferase , fibroblast , transfection , microbiology and biotechnology , reporter gene , dermal fibroblast , biology , plasmid , promoter , gene expression , cell culture , gene , biochemistry , genetics
A cell‐based assay system for monitoring COL1A2 [α2(I) collagen gene] promoter activity was developed to determine the influence of activated COL1A2 promoter in human dermal fibroblast cells. A pLuc‐ COL1A2 promoter plasmid that expresses the luciferase reporter gene in response to COL1A2 promoter activity was constructed. The pLuc‐ COL1A2 promoter plasmid and pCI‐neo plasmid containing the NPT (neomycin phosphotransferase) gene for Geneticin resistance in host cells were co‐transfected into human dermal fibroblast cells. COL1A2 promoter activities were measured by luciferase reporter gene assay using a luminescence detection method. Fibroblast cell transfectants treated with TNFα (tumour necrosis factor α), known to be an inhibitor of COL1A2 promoter expression, showed a reduction of COL1A2 promoter activity in a concentration‐dependent manner, whereas TGF‐β (transforming growth factor‐β), known to be a stimulator of COL1A2 promoter expression, increased COL1A2 activity in a concentration‐dependent manner. This assay system could be used to quantitatively measure COL1A2 promoter activity in human dermal fibroblast cells and allow the screening of anti‐wrinkle agents from various synthetic chemicals and natural products.