Project description:Pluripotent marker correlation between miR-302/367-iPS, ES and fibroblast cells Mouse fibroblast were reprogrammed with miR-302/367 lentiviral vector, total RNA was extracted by trizol and microarray assay was performed
Project description:To define the role of miR-302-367 cluster in cardiac development, we overexpressed miR-302-367 cluster in mouse heart by using R26R-miR-302-367; Nkx2.5-Cre mice. This data set contains the microarrays examining gene expression in the hearts of R26R-miR-302-367; Nkx2.5-Cre mice at postnatal day 14.
Project description:To define the role of miR-302-367 cluster in cardiac development, we overexpressed miR-302-367 cluster in mouse heart by using R26R-miR-302-367; Nkx2.5-Cre mice. This data set contains the microarrays examining gene expression in the hearts of R26R-miR-302-367; Nkx2.5-Cre mice at postnatal day 14. We overexpressed miR-302-367 cluster in developing mouse heart using Nkx2.5-Cre mouse line
Project description:Malignant germ-cell-tumours (GCTs) are characterised by microRNA (miRNA/miR-) dysregulation, with universal over-expression of miR-371~373 and miR-302/367 clusters regardless of patient age, tumour site, or subtype (seminoma/yolk-sac-tumour/embryonal carcinoma). These miRNAs are released into the bloodstream, presumed within extracellular-vesicles (EVs) and represent promising biomarkers. Here, we comprehensively examined the role of EVs, and their miRNA cargo, on (fibroblast/endothelial/macrophage) cells representative of the testicular GCT (TGCT) tumour microenvironment (TME). Small RNA next-generation-sequencing was performed on 34 samples, comprising representative malignant GCT cell lines/EVs and controls (testis fibroblast [Hs1.Tes] cell-line/EVs and testis/ovary samples). TME cells received TGCT co-culture, TGCT-derived EVs, and a miRNA overexpression system (miR-371a-OE) to assess functional relevance. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were overexpressed in all TGCT cells/subtypes compared with control cells and were highly abundant in TGCT-derived EVs, with miR-371a-3p/miR-371a-5p the most abundant. TGCT co-culture resulted in increased levels of miRNAs from the miR-371~373 and miR-302/367 clusters in TME (fibroblast) cells. Next, fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME (fibroblast/endothelial/macrophage) cells. TME (fibroblast/endothelial) cell treatment with EVs derived from different TGCT subtypes resulted in increased miR-371~373 and miR-302/367 miRNA levels, and other generic (eg, miR-205-5p/miR-148-3p) and subtype-specific (seminoma, eg, miR-203a-3p; yolk-sac-tumour, eg, miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), via direct mRNA downregulation and alteration of relevant pathways. TGCT cells communicate with nontumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, potentially contributing to tumour progression.
Project description:We report the generation and characterization of DICER1-deficient hESCs. We uncover an unexpected requirement for DICER1 as well as essential pro-survival roles of members of the mir-302- 367 and mir-371- 373 clusters in hESCs. Our work provides a robust platform for interrogating microRNA function in hESC and differentiation.
Project description:Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSC). Forced expression of transcription factors might lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSC. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can potentially reduce this heterogeneity. Here we describe a high efficiency reprogramming of human somatic cells into iPSC using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells with more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high quality reprogramming of human iPSC.
Project description:Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSC). Forced expression of transcription factors might lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSC. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can potentially reduce this heterogeneity. Here we describe a high efficiency reprogramming of human somatic cells into iPSC using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells with more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high quality reprogramming of human iPSC.
Project description:We collected whole genome testis expression data from hybrid zone mice. We integrated GWAS mapping of testis expression traits and low testis weight to gain insight into the genetic basis of hybrid male sterility.