Effect of Trp53 mutations on mouse oviductal epithelial cell spheroids
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA946636 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24313400.fastq.gz | Fastqsanger.gz | |||
| SRR24313401.fastq.gz | Fastqsanger.gz | |||
| SRR24313402.fastq.gz | Fastqsanger.gz | |||
| SRR24313403.fastq.gz | Fastqsanger.gz | |||
| SRR24313404.fastq.gz | Fastqsanger.gz |
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Project description:To identify mutant p53-dependent pathway alterations that are enhancing spheroid survival properties in mouse oviductal epithelial (OVE) cell lines, we generated OVE4 and OVE16 cell lines with either Trp53 deletion or stable expression of p53R175H. RNA-seq was performed on RNA from OVE spheroids to determine differential gene expression. Gene set enrichment analysis was performed to indentify enriched pathways in parental OVE spheroids compared to spheroids with Trp53 deletion or expression of p53R175H
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Project description:We established murine fallopian tube epithelial organoids from a B6J.129(B6N)-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J mouse. Subsequently, we knocked out Trp53 by introducing sgRNA into the organoids, nutlin-3 selection, and single-organoid cloning. The Trp53-knocked organoids grew faster than the normal organoids. We also analyzed the transcriptomic differences caused by Trp53-knockout by RNA-sequencing and gene set enrichment analysis (GSEA), which indicated that Trp53-knockout reduced cilium-related gene expression. In human HGSC precancerous lesions, such as serous tubal intraepithelial carcinoma (STIC), differentiation to ciliated cells has been reported to be down-regulated; therefore, the genetic manipulation was supposed to mimic the process of carcinogenesis of HGSC.
2024-05-07 | GSE255128 | GEO
Project description:The objective was to determine the transcriptional effect of IL-17A on primary colonic epithelial cells in various differentiation states in vitro. The three states included 1) stem/progenitor cell spheroids grown in 50% L-WRN media as described in our previous publication (PMID: 24232249), 2) differentiating colonic epithelial spheroids (DM) placed in differentiation media without L-WRN for 24 hours, 3) terminally differentiated colonocyte spheroids placed in differentiation media without L-WRN for 48 hours as described in our previous publication (PMID: 27264604). Each condition was cultured with or without 20ng/ml recombinant mouse IL-17A for the final 24 hours.
2018-04-10 | E-MTAB-6365 | biostudies-arrayexpress
Project description:Purpose: Transcriptome profiling of organoids/tissues derived from murine oviduct and OSE was performed to study and compare the gene expression profiles in the both suspected origins of HG-SOC. CRISPR-Cas9 modified oviductal organoids (TBP clones, which were mutated in Trp53, Brca1 and Pten), the tumors derived upon xenotransplantation of the TBP-clones and subsequent tumor-derived organoids were additionally profiled to show the transcriptional change upon aquiring mutations and tumor development. Methods: Both oviductal and OSE organoids as well as oviductal TBP-clones and tumor-derived organoids were grown in Wnt-conditioned media prior harvesting for RNA extraction. Additionally, RNA was extracted from healthy oviductal and OSE tissues and Ovi-TBP-clone-derived tumor tissues. RNA was converted to cDNA and libraries were prepared using the CelSeq2 method and sequenced. Samples were sequenced on Illumina NextSeq500 by using 75-bp paired-end sequencing. Paired-end reads from Illumina sequencing were aligned to the mouse genome (GRCm38 assembly) by BWA. DESeq2 (v1.18.0) package was used for read normalization and differential expression analysis. Gene set enrichment analysis (GSEA) was performed using gene lists for motile cilium assembly against normalized RNA-seq reads of healthy oviductal an OSE organoids using GSEA software v3.0 beta2. GSEA was additionally used to detect enrichment of signature gene sets of different HG-SOC subtypes in the TBP-clone-derived murine tumors Results: Transcriptome analysis of oviductal and OSE organoids/tissues revealed tissue-specific gene expression pattern in the organoid systems. In contrast to OSE counterparts, oviductal organoids showed enrichment in motile cilium assembly genes, reflecting the presence of ciliated cells in this model. Results: Oviductal TBP-clones gave rise to different tumor phenotypes with distinct gene expression profiles. Analysis of the transcriptomes of these tumors divided the TBP-clone-derived tumors into two distinct HG-SOC molecular subtypes (differentiated- or immunoreactive-like subtypes), confirming the potential of organoids in modeling HG-SOC. Results: The tumor-derived organoids retained the distinct phenotypes observed in the originating Ovi-TBP-clone derived tumors. Subset of tumors showed epithelial and others mesenchymal features that were maintained in the respective organoid cultures also in the gene expression level. Conclusions: RNA sequencing showed tissue specific differences in murine oviductal and OSE organoids, and revealed HG-SOC-like features of oviductal mutants.
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Project description:More than 60% of pregnancy loss occurs during the first trimester. As we lack a complete understanding of the environment within the oviduct (Fallopian tube in humans) and its role in regulating early pregnancy, it is difficult to provide diagnostic tools for oviductal origin of early pregnancy loss. To determine the role of progesterone (P4), we conditionally ablated the classical P4 receptor (Pgr) in oviductal epithelial cells using the Wnt7aCre/+;Pgrf/f (hereafter Pgrd/d) mouse model. Approximately 40% of embryos collected from Pgrd/d females at 3.5 days post-coitus were nonviable or developmentally delayed suggesting that epithelial PGR expression in the oviduct is required for normal embryonic development. Single-cell RNA-sequencing analysis showed an upregulation of genes involved in proinflammatory pathway (i.e., interleukin 22 (IL-22) and genes in IL-22 signaling) in secretory epithelial cells of Pgrd/d females as compared to controls. A loss of PGR in the epithelial cells resulted in an increase immune cell recruitment into the oviduct as compared to controls. Conversely, ovariectomized wild-type (WT) females treated with exogenous P4 showed a reduced expression of inflammatory response genes. Pharmacological inhibition of inflammation using non-steroidal anti-inflammatory drugs significantly reduced IL-22 levels in the oviducts and rescued embryo developmental rates in Pgrd/d females. Co-culture of WT zygotes with IL-22 significantly decreased the number of expanded blastocysts. Our findings indicate that P4 signaling through the classical PGR in oviductal epithelial cells is required for the immunoregulatory function and is indispensable for normal preimplantation embryonic development. These data may contribute to an understanding of the mechanisms underlying pre-implantation pregnancy failure, which can inform the development of targeted diagnostic tools and therapeutic interventions to improve reproductive outcomes when embryonic aneuploidy is not present.
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Project description:Intestinal epithelial spheroids were isolated from mouse jejunum as described in our previous publication (Miyoshi and Stappenbeck. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nature Protocols. 2013. PMID: 24232249) and then grown in 50% L-WRN conditioned medium (for stem cells) or differentiation medium containing dmPGE2 (for wound-associated epithelial cells) or differentiation medium containing EP4 inhibitor (for enterocytes). Transcription profiling was performed to gain insight into the characteristics distinguishing these cell types.
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