Project description:To understand the function of MSI1 in pluripotent stem cells, RNA-seq assays were performed on mouse embryonic stem cells R1, MSI1 knockout cell line R1-C5, human embryonic stem cells H9, RRM knockout cell line H9-C8, MSI1 full-length overexpression cell line H9-MSI1OE, MSI1C variant overexpression cell line H9-MSI1 (138-362) OE , H9-MSI1(272-362)OE. RNA bound by MSI1 in R1 and H9, and MSI1C variants MSI1 (138-362), MSI1(272-362) were detected using RIP-seq.
Project description:RIP-chip analysis to identify mRNA preferentially associated with Msi1 protein. RIP-Chip experiments were performed on two biologically replicated samples transfected with the BAP-Msi1 construct and a control sample from cells transfected with the BAP-Control construct. A total of 8 microarrays were carried on using technical replicates of BAP-Msi1 vs. BAP-Control for each dye orientation.
Project description:Musashi1 (Msi1) is a highly conserved RNA binding protein that is required during the development of the nervous system. Msi1 has a role in neural stem cells, controlling the balance between self-renewal and differentiation. Msi1 has also been implicated in cancer, being highly expressed in multiple tumor types. In this study, we analyzed Msi1 expression in a large cohort of medulloblastoma samples and showed that Msi1 is highly expressed in tumor tissue compared to normal cerebellum and that high Msi1 expression is associated with a poor prognosis. Using a nude mouse xenograft model, we demonstrate that Msi1 is important for tumor growth. We then used RIP-chip (ribonucleoprotein immunoprecipitation followed by microarray analysis) to identify mRNA targets of Msi1 in medulloblastoma. In conclusion, our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target. RIP-Chip analysis to identify mRNA preferentially associated with Msi1 protein. RIP-Chip experiments were performed on two biologically replicated samples. A total of 8 microarrays were carried on using technical replicates of Msi1 antibody vs. prebleed serum for each dye orientation. We prepared two biological replicates for two different arrays. Each array consisted of 4 microarrays with 2 replicates for each dye orientation.
Project description:Glioma stem cells (GSCs) have been identified in glioma tissues and suggested to play important roles in the tumorigenesis of glioblastoma multiform (GBM). We established a novel cellular bioinformative pipeline that consisted of principal component analysis (PCA) with factor loading, intracellular pathway analysis, and immunopathway analysis and attempted to clarify the differences in gene expression profiles comprehensively among GSCs, a glioma cell line (U251), and a human GBM tissue (hGBM). To this end, we extracted total RNAs from the GSCs, U251, and the hGBM, performed microarray, and applied the data to the bioinformatics analyses described above. As the results, PCA clearly distinguished the three groups. Moreover, the second principal component (PC2) distinguished the GSCs from the hGBM and U251; it reflected the characteristics of stemness. The factor loading for PC2 suggested MYCN, DPP4, and, MIF as contributing factors to the stemness of GSCs. We clarify the similarities and differences among samples such as the GSCs, U251, and hGBM.
Project description:mRNA-seq and ribosome profiling of neural stem cells overexpressing or knocked out for Musashi RNA-binding proteins Study of the global effects of Musashi (Msi) proteins on the transcriptome of embryonic neural stem cells. Neural stem cells were derived from brains of E12.5 or E13.5 embryos engineered to have inducible Msi1 or Msi2 genes, or from embryos with double floxed alleles of Msi1 and Msi2 carrying a Tamoxifen-induclble Cre (CreER). The overexpression mice were made using the Flp-in system (OpenBioSystems), where a cDNA of interest (in this case Msi1 or Msi2) is knocked into the Collagen (Col1A1) locus. The expression of the cDNA of interest is driven by m2rTTA that is knocked into the Rosa26 locus (R26). KH2 describes a strain containing the R26-m2rTTA but lacking Msi1 or Msi2 cDNA. MSI1 describes a strain containing R26-m2rTTA and Msi1 cDNA in Col1A1. MSI2 describes a strain containing R26-m2rTTA and Msi2 cDNA in Col1A1. C1 describes a strain lacking the CreER allele but containing double floxed alleles of Msi1/Msi2 (used as Tamoxifen control). C4 describes a strain carrying the CreER allele and double floxed alleles of Msi1/Msi2.