Reporter gene assay against lipophilic chemicals based on site-specific genomic recombination of a nuclear receptor gene, its response element, and a luciferase reporter gene within a stable HeLa cell line.
Genomic recombination was performed in a genetically modified stable HeLa cell line, HeLa55, using a uniquely designed donor vector harboring an exchange cassette comprised of the human glucocorticoid receptor (hGR) gene, its response element, and a luciferase reporter gene, to generate stable hGRLuc clones. After screening for cassette insertion, the selected stable clone, hGRLuc-7, showed high integration stability of the exchange cassette over 20 passages with significantly high luciferase activity and fold inductions of up to 40- to 50-fold. In addition, the cells were evaluated with synthetic glucocorticoid, dexamethasone, and a reasonable EC(50) value of approximately 2.3 x 10(-9) M was obtained. Strong and weak agonists, non-responsive chemicals, and hGR antagonists were also evaluated in which the stable hGRLuc-7 clone showed both high sensitivity and selectivity. The technology presented in this work is simple and reproducible, and shows great potential for the future development of genetically modified stable cell systems which are applicable in both fundamental and application researches of nuclear receptors.
Mori
Tetsushi
T
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
Saito
Fumiyo
F
Yoshino
Tomoko
T
Takeyama
Haruko
H
Matsunaga
Tadashi
T
D001681:Biological Assay / Q000379:methods*; D003907:Dexamethasone / Q000008:administration & dosage*; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000008:administration & dosage; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism / Q000187:drug effects*; D008156:Luciferases / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics; D020218:Response Elements / Q000235:genetics
D005938:Glucocorticoids; D018160:Receptors, Cytoplasmic and Nuclear; D003907:Dexamethasone; D008156:Luciferases