Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene expression changes in the intestinal crypts of mice with loss of imprinting

ABSTRACT: To identify genes differentially expressed in the intestinal crypts between LOI(+) and LOI(-) mice, we performed microarray experiments using RNA extracted from laser capture microdissection, by microdissecting an average of 8,000 crypts from each of 3 LOI(+) and 3 LOI(-) mice. Keywords: genetic modification design H19 mutant mice with C57BL/6J background carrying a deletion in the H19 gene (3 kb) and 10 kb of the upstream region including differentially methylated region (DMR) were maintained without LOI phenotype by breeding female wild-type C57BL/6J and male H19+/-. Experimental crosses were performed between female H19+/- and male wild-type C57BL/6J to obtain LOI(+) mice and LOI(-) mice. Igf2 on the maternal allele is silenced in LOI(-), i.e. wild type, mice. But when H19 DMR deletion is inherited from mother, the normally silenced Igf2 on maternal allele is activated and LOI (loss of imprinting) of Igf2 occurs. LOI(+) and LOI(-) mice were sacrificed at 120 days, and the tissue pieces of the small intestine were collected from the middle (the third fifth) part and kept frozen at -80C. To detect gene expression change in intestinal progenitor cells clearly, Laser Capture Microdissection (LCM) was performed to collect intestinal crypt cells. The 10 um thick sections were prepared from the frozen small intestine pieces, and 8,000 intestinal crypts at average were dissected by LCM for each of three LOI(+) and LOI(-) mice, and 2 ug of total RNA at least were collected using RNeasy Kit (GIAGEN). 1.7 ug of total RNA for each sample were used for labeling to compare gene expressions in intestinal crypt between LOI(+) and LOI(-) mice.

ORGANISM(S): Mus musculus

SUBMITTER: Patrick Onyango

PROVIDER: E-GEOD-8583 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk.

Kaneda Atsushi A Wang Chiaochun J CJ Cheong Raymond R Timp Winston W Onyango Patrick P Wen Bo B Iacobuzio-Donahue Christine A CA Ohlsson Rolf R Andraos Rita R Pearson Mark A MA Sharov Alexei A AA Longo Dan L DL Ko Minoru S H MS Levchenko Andre A Feinberg Andrew P AP

Proceedings of the National Academy of Sciences of the United States of America 20071217 52

Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II ...[more]

PMID: 18087038

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Project description:Abstract: Dysregulation of the imprinted H19/IGF2 locus can lead to Silver-Russell Syndrome (SRS) in humans. However, the mechanism of how abnormal H19/IGF2 expression contributes to various SRS phenotypes remains unclear, largely due to incomplete understanding of the developmental functions of these two genes. We previously generated a mouse model with humanized H19/IGF2 ICR (hIC1) on the paternal allele that exhibited H19/Igf2 dysregulation together with SRS-like growth restriction and perinatal lethality. Here we dissect the role of H19 and Igf2 in cardiac and placental development utilizing multiple mouse models with varying levels of H19 and Igf2. We report severe cardiac defects such as ventricular septal defects (VSDs) and thinned myocardium, placental anomalies including thrombosis and vascular malformations, together with growth restriction in mouse embryos that correlated with the extent of H19/Igf2 dysregulation. Transcriptomic analysis using cardiac endothelial cells of these mouse models shows that H19/Igf2 dysregulation disrupts pathways related to extracellular matrix (ECM) and proliferation of endothelial cells. Our work links the heart and placenta through regulation by H19 and Igf2, demonstrating that accurate dosage of both H19 and Igf2 is critical for normal embryonic development, especially related to the cardiac-placental axis. Methods: E12.5 hearts were lysed with Collagenase, Dispase II, and DNase I. Cardiac endothelial cells were collected using MACS CD31 microbeads and RNA was isolated using RNeasy Micro kit. After confirming RNA integrity using Bioanalyzer, mRNA library was generated from 25ng RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module and Ultra II RNA Library Prep Kit. Library quality was assessed by Bioanalyzer and TapeStation. Sequencing was performed on NovaSeq 6000. Quality of raw fastq reads was assessed using FastQC version 0.11.5. Reads were aligned to the GRCm38/mm10 reference using STAR version 2.4.0i with default parameters and maximum fragment size of 2000 bp. Properly paired primary alignments were retained for downstream analysis using Samtools version 1.9. Count matrices were generated using FeatureCounts version 1.6.2 against RefSeq gene annotation and read into DESeq2 to perform normalization and statistical analysis.

Dataset Information

Gene expression changes in the intestinal crypts of mice with loss of imprinting

Publications

Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk.

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