title: Differential display system with vertebrate-common degenerate oligonucleotide primers: uncovering genes responsive to dioxin in avian embryonic liver.
abstract: To assess possible impacts of environmental pollutants on gene expression profiles in a variety of organisms, we developed a novel differential display system with primer sets that are common in seven vertebrate species, based on degenerate oligonucleotide-primed PCR (DOP-PCR). An 8-mer inverse repeat motif was found in most transcripts from the seven vertebrates including fish to primates with detailed transcriptome information; more than 10,000 motifs were recognized in common in the transcripts of the seven species. Among them, we selected 275 common motifs that cover about 40-70% of transcripts throughout these species, and designed 275 DOP-PCR primers that were common to seven vertebrate species (common DOP-PCR primers). To detect genes responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing embryos, differential display with common DOP-PCR primers was applied to embryonic liver of two avian species, the chicken (Gallus gallus) and the common cormorant (Phalacrocorax carbo), which were exposed in ovo to TCDD. The cDNA bands that showed differences between the control and TCDD-treated groups were sequenced and the mRNA expression levels were confirmed by real-time RT-PCR. This approach succeeded in isolating novel dioxin-responsive genes that include 10 coding genes in the chicken, and 1 coding gene and 1 unknown transcript in the cormorant, together with cytochrome P450 1As that have already been well established as dioxin markers. These results highlighted the usefulness of systematically designed novel differential display systems to search genes responsive to chemicals in vertebrates, including wild species, for which transcriptome information is not available.
authors.affiliation: School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Teraoka
authors.forename: Shino
authors.initials: S
authors.lastname: Ito
authors.forename: Haruki
authors.initials: H
authors.lastname: Ikeda
authors.forename: Akira
authors.initials: A
authors.lastname: Kubota
authors.forename: M M
authors.initials: MM
authors.lastname: Abou Elmagd
authors.forename: Takio
authors.initials: T
authors.lastname: Kitazawa
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Kim
authors.forename: Hisato
authors.initials: H
authors.lastname: Iwata
authors.forename: Daiji
authors.initials: D
authors.lastname: Endoh
mesh_terms: D000818:Animals; D001483:Base Sequence; D001717:Birds / Q000196:embryology* / Q000235:genetics*; D002642:Chick Embryo; D002645:Chickens; D017931:DNA Primers / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D055029:Inverted Repeat Sequences / Q000235:genetics; D008099:Liver / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger