Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector.
We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3'-l-triiodothyronine (T(3)) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T(3)-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T(3) that significantly induced the luciferase gene expression was 10(-11)M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10(-6)M, had a weak T(3)-agonist activity, whereas trichlorobisphenol A, at 10(-8) - 10(-6)M had a weak T(3)-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T(3)-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals.
Sugiyama
Shin-Ichiro
S
Department of Biology, Faculty of Science, Shizuoka University, Japan.
Miyoshi
Hiroyuki
H
Yamauchi
Kiyoshi
K
D000818:Animals; D001559:Benzhydryl Compounds; D002460:Cell Line; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010636:Phenols / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D020218:Response Elements / Q000235:genetics*; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology*; D014982:Xenopus laevis; D000098826:Bisphenol A Compounds
D001559:Benzhydryl Compounds; D049452:Green Fluorescent Proteins; D008156:Luciferases; D010636:Phenols; D011993:Recombinant Fusion Proteins; D014284:Triiodothyronine; C485184:enhanced green fluorescent protein; C006780:bisphenol A; D000098826:Bisphenol A Compounds