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Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs.

ABSTRACT: To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO) and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524). The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015].

SUBMITTER: Liu M

PROVIDER: S-EPMC4583647 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs.

Liu Ming M Wang Xiu-Ping XP

Genomics data 20150604

To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO) and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE ...[more]

PMID: 26484260

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Project description: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 ÂµM TCDD. Experiment 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 Âµm; Corning Inc., New York] placed on a stainless steel grid. The basal culture medium was Dulbeccoâs modified Eagleâs 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 JyvÃ¤skylÃ¤, JyvÃ¤skylÃ¤, Finland) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 Âµg/ml. TCDD at the concentration of 1 ÂµM, 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 ÂºC 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, 15â29 explants were included in each experimental group. Experiment Overall Design: Microarray analysis: Experiment Overall Design: 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 37ÂºC 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 Âµg of labelled cRNA was fragmented by incubation at 94 ÂºC 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 ÂºC, 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.

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Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs.

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Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs.

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