TY - JOUR
T1 - 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
AU - Mori, Tetsushi
AU - Saito, Fumiyo
AU - Yoshino, Tomoko
AU - Takeyama, Haruko
AU - Matsunaga, Tadashi
PY - 2008/4/15
Y1 - 2008/4/15
N2 - 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.
AB - 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.
KW - Genomic recombination
KW - Human glucocorticoid receptor
KW - Lipophilic chemicals
KW - Stable cell reporter gene assay
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U2 - 10.1002/bit.21729
DO - 10.1002/bit.21729
M3 - Article
C2 - 18023048
AN - SCOPUS:41049118497
VL - 99
SP - 1453
EP - 1461
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 6
ER -