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

Tetsushi Mori, Fumiyo Saito, Tomoko Yoshino, Haruko Takeyama, Tadashi Matsunaga

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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 EC50 value of approximately 2.3 × 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.

Original languageEnglish
Pages (from-to)1453-1461
Number of pages9
JournalBiotechnology and bioengineering
Volume99
Issue number6
DOIs
Publication statusPublished - 2008 Apr 15
Externally publishedYes

Keywords

  • Genomic recombination
  • Human glucocorticoid receptor
  • Lipophilic chemicals
  • Stable cell reporter gene assay

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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