Unknown,Transcriptomics,Genomics,Proteomics

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Novel genes involved in the pathophysiology of gonadotropin-dependent adrenocortical tumorigenesis in mice.

ABSTRACT: The inh-alpha/Tag mouse is a murine adrenocortical tumor model that combines gonadotropin-dependent tumorigenesis with SV40Tag expression (which inhibits p53). These mice have been extensively used to study adrenocortical and gonadal tumorigenesis, as they initially present with gonadal tumors. To induce adrenocortical tumorigenesis, the inh-alpha/Tag mice need to be prebubertally gonadectomized. Generally, females develop adrenal tumors faster and larger in size than males, with a very high individual variations of tumor volume and histological appearance. In this study, we used the inh-alpha/Tag mouse model to analyze the molecular basis of the LH-dependent adrenal tumorigenesis. Experimental inh-alpha/Tag and control littermates (C57Bl/6N) mice of both sexes were used as the animal model. inh-alpha/Tag and WT mice were gonadectomized at 21-24 days of age. Mice were sacrificed at the age of 30 weeks (+/-1 week) and adrenals snap-frozen in liquid nitrogen for microarray analysis. Additionally, a group of 27 week-old inh-alpha/Tag mice (+/-1 week) were treated for 3 weeks with GnRH antagonist cetrorelix acetate (#C5249, Sigma-Aldrich) (3mg/kg/48h). Mice were sacrificed at the age of 30 weeks (+/-1 week) and adrenals snap-frozen in liquid nitrogen for microarray analysis.

ORGANISM(S): Mus musculus

SUBMITTER: Kirstine Belling

PROVIDER: E-MTAB-5310 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We sought to determine which gene transcripts are enriched in Wnt-responsive adrenocortical mouse cells compared to the entire adrenocortical mouse cell population in vivo. To this end, we employed transgenic reporter mice that label Wnt-responsive cells with GFP expression (TCF/Lef:H2B-GFP mice) or label all adrenocortical cells with GFP expression (Sf1:eGFP mice). GFP-positive adrenocortical cells were obtained from 6-week-old male TCF/Lef:H2B-GFP mice and Sf1:eGFP mice independently. 10 adrenals per genotype per sort were minced and digested by incubation in DMEM:F12 containing 0.1% collagenase/ 0.01% DNaseI solution for 1 h at 37Â°C. A single cell suspension was obtained following mechanical dispersion, filtration through a 40 micron nylon cell strainer, centrifugation at 1500rpm for 5 min followed by re-suspension in sterile 1X PBS containing 10% cosmic calf serum and 10Î¼g/mL Propidium iodide. 10,000-50,000 viable GFP-positive cells were isolated via FACS using a BD FACSAria III cell sorter. RNA was extracted using an RNeasy Micro Kit (Qiagen) from 4 independent sorts per genotype. cDNA were prepared according to the NuGen WT-Pico V2 kit protocol from 5 ng total RNA (Ovation PicoSL WTA System V2 P/N 3312). Biotinylated single-stranded cDNA were prepared from 3ug of cDNA (Encore Biotin Module P/N 4200-12, 4200-60, 4200-A01). Targets were assayed on the Mouse Gene ST 1.1 strip arrays using the Affymetrix Gene Atlas system (software version 1.0.4.267). One TCF/Lef:H2B-GFP array was deemed low-quality and discarded. Two-sample T-tests were used to compare the two groups of samples. We also supply a supplementary file holding the data and some statistical analysis, as well as probe-set annotation that we used at that time (users may wish to obtain new annotation though). We analyzed only 28944 probe-sets with category "main", "---", and "flmrna->unmapped" according to Affymetrix annotation. GFP-positive adrenocortical cells were obtained from 6-week-old male TCF/Lef:H2B-GFP mice and Sf1:eGFP mice independently. 10 adrenals per genotype per sort were minced and digested by incubation in DMEM:F12 containing 0.1% collagenase/ 0.01% DNaseI solution for 1 h at 37Â°C. A single cell suspension was obtained following mechanical dispersion, filtration through a 40 micron nylon cell strainer, centrifugation at 1500rpm for 5 min followed by re-suspension in sterile 1X PBS containing 10% cosmic calf serum and 10Î¼g/mL Propidium iodide. 10,000-50,000 viable GFP-positive cells were isolated via FACS using a BD FACSAria III cell sorter. RNA was extracted using an RNeasy Micro Kit (Qiagen) from 4 independent sorts per genoytpe. cDNA were prepared according to the NuGen WT-Pico V2 kit protocol from 5 ng total RNA (Ovation PicoSL WTA System V2 P/N 3312). Biotinylated single-stranded cDNA were prepared from 3ug of cDNA (Encore Biotin Module P/N 4200-12, 4200-60, 4200-A01). Targets were assayed on the Mouse Gene ST 1.1 strip arrays using the Affymetrix Gene Atlas system (software version 1.0.4.267).

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Novel genes involved in the pathophysiology of gonadotropin-dependent adrenocortical tumorigenesis in mice.

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