BioProject

Dioxins are ubiquitous environmental poisons that are developmentally toxic. More...

Dioxins are ubiquitous environmental poisons that are developmentally toxic. Exposure of mouse E14 tooth germs to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to reduced tooth size and deformation of cuspal morphology implying the induction of a variety of biological responses on both cellular and molecular levels. To verify such responses at the gene level, mouse embryonic cap staged tooth germs were cultured for 24 h with/without 1 µM TCDD. Keywords: tissue culture, exposure to TCDD, tooth development Overall design: Organ culture: Mandibular molar tooth regions from E14 mouse embryos (NMRI X NMRI; the day of vaginal plug = E0) were dissected under a stereomicroscope and transferred to an organ culture. The tissue explants were supported by polycarbonate Nuclepore filters [pore size, 0.1 µm; Corning Inc., New York] placed on a stainless steel grid. The basal culture medium was Dulbecco’s modified Eagle’s medium (DMEM) containing Glutamax-1 (Gibco BRL, Paisley, Scotland) supplemented with 10% fetal calf serum (FCS; Gibco BRL). TCDD (from the Department of Chemistry, University of Jyväskylä, Jyväskylä, Finland) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 µg/ml. TCDD at the concentration of 1 µM, or DMSO, was added to the basal culture medium from the start of culture. The culture dishes were kept in a humidified incubator at 37 ºC in an atmosphere of 5% CO2. The tissue explants were cultured for 24 hours, after which they were snap frozen in liquid nitrogen. Explants from several experiments were collected and kept in liquid nitrogen. In a single experiment, 15–29 explants were included in each experimental group. Microarray analysis: Three separate experiments were combined for each replicate sample. About 60 explants each were pooled and homogenized while still frozen in liquid nitrogen. Total RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the total RNA by using a GeneChip T7-Oligo(dT)24 Promoter Primer Kit (Affymetrix) and Superscript II Choice System (Invitrogen Life Sciences). The double stranded cDNA was cleaned-up with the GeneChip Sample Cleanup Module (Affymetrix). Biotin-labelled cRNA was synthesized by in vitro transcription usin a RNA labelling kit from Enzo Life Sciences, Inc. The labelling time was 5 h in 37ºC waterbath. All syntheses were done according to the manufacturers instructions. Unincorporated NTPs were removed with the GeneChip Sample Cleanup kit and the concentration of the labelled cRNA was mesured by spectrophotometric analysis and quantificated. 15 or 16 µg of labelled cRNA was fragmented by incubation at 94 ºC for 35 min in the fragmentation buffer supplied in the GeneChip Sample Cleanup kit. The cRNA was hybridized to a murine U74v2A array (Affymetrix) for 16 h at 45 ºC, washed and stained with streptavidine-phycoerythrin. The arrays were scanned in a Genechip System confocal scanner (Aligent) and Affymetrix Microarray Suite 5.0 was used to scan and analyze the relative abundance of each gene. The target signal scaling was set to 100. Less...