Allele-specific expression profiling of imprinted genes in mouse isogenic pluripotent tissues and cell lines
Ontology highlight
ABSTRACT: Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we perform allele-specific RNA-Seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic stages and in pluripotent cell lines. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos or from embryos obtained after nuclear transfer (NT), as well as B6D2F1 ESCs and EpiSCs derived after parthenogenetic activation (PGA). Notably, the PGA-derived cell lines contain a mosaic genotype due to genomic recombination occurring in the parental B6D2F1 oocyte during meiosis. As the homozygous genomic regions of the PGA-derived cells are not compatible with allele-specific RNA-Seq, we developed an RNA-Seq based genotyping strategy that allows identification of the informative heterozygous regions. Global imprinting analysis shows that ESC lines largely lose imprints as compared to their corresponding embryonic tissues. Fertilized EpiSC and EpiSC-NT lines generally maintain imprinted gene expression. However, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. EpiSC-PGA lines display loss of imprinting, with known paternally-expressed genes being silenced and known maternally-expressed genes consistently showing doubled expression. Interestingly, most female EpiSC lines show monoallelic expression of Xist and full skewing of X inactivation, suggesting a (near) clonal origin. Together, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential.
Project description:Genome wide comparison of gene expression between EpiSC lines derived from fertilized (FT) embryos and somatic cell nuclear transfer (NT) embryos. EpiSC lines were derived from fertilized and somatic cell nuclear transfer embryos and cultured until 15 to 20 passages. RNA was then extracted in order to compare transcriptomic profiles.
Project description:Comparison of ES cell line of different genetic background and different cloning methods to identify changes on gene expression levels between these ES cell lines. The main question behind the experiments id, if there is major/important difference on gene expression level between NT embryo-derived ES cell liens and control embryo derived ES cell lines. Ih has an effect on therapeutic cloning that means the nuclei donor cell source could be an important question. We also included different genetic background cell lines (HM-1 is 129/SV, #4 ES cell line is B6D2F1 derived heterozygote cell line) to compare the effect of gentic background differences. Hetero- and homozygosis con be compared based on the PGA ES cell line from the same genetic backgound. Keywords: cDNA microarray, murine ES cells, nucleous transfer
Project description:To characterize molecular features of new EpiSC lines established by the Wnt inhibition method, global gene expression profiles of the EpiSC lines were determined by microarray, and compared to those of EpiSC lines established by other group using the conventional method. Epiblasts, the source of the EpiSCs, and mESCs were also analyzed.
Project description:The requirements for self-renewal differ between EpiSCs and ES cells and the underlying mechanism is largely unknown. Here we show that mouse EpiSCs can be efficiently derived and robustly propagated even from single cells, using two small-molecule inhibitors: CHIR99021 and XAV939. The whole-genome microarray analyses is performed to confirm the identity of EpiSC maintained in CHIR/XAV by comparing the expression profile in EpiSC-CHIR/XAV to those in ESC maintained in 2i and EpiSC maintained in FGF2/activin.
Project description:Epiblast stem cells (EpiSCs) were derived from the epiblast or the ectoderm (epi/ect) of pre-gastrula stage to late-bud stage mouse embryos. To identify if the EpiSCs retain any original stage specific characteristics or which developmental stage of epi/ect they most closely related to, we performed microarray analysis to compare the gene expression profile of multiple EpiSC lines with that of epi/ect of 7 different stages.
Project description:To characterize molecular features of new EpiSC lines established by the Wnt inhibition method, global gene expression profiles of the EpiSC lines were determined by microarray, and compared to those of EpiSC lines established by other group using the conventional method. Epiblasts, the source of the EpiSCs, and mESCs were also analyzed. EpiSCs and mESC were maintained as undifferentiated state on feeder layers. The stem cells were then separated from feeders, and RNAs were extracted from the stem cell lines. Embryonic tissues were manually dissected out from mouse embryos of E5.5, E6.5 or E7.5, from which RNAs were extracted.
Project description:The requirements for self-renewal differ between EpiSCs and ES cells and the underlying mechanism is largely unknown. Here we show that mouse EpiSCs can be efficiently derived and robustly propagated even from single cells, using two small-molecule inhibitors: CHIR99021 and XAV939. The whole-genome microarray analyses is performed to confirm the identity of EpiSC maintained in CHIR/XAV by comparing the expression profile in EpiSC-CHIR/XAV to those in ESC maintained in 2i and EpiSC maintained in FGF2/activin. Total RNA from ESC-2i, EpiSC-CHIR/XAV, and EpiSC-FGF2/activin were extracted for microarray analisys
Project description:Parthenogenetic embryonic stem cells (PESCs) may have future utility in cell replacement therapies. We examined genome-wide mRNA expression profiles of monkey PESCs relative to ESCs derived from fertilized embryos. Several known paternally-imprinted genes were in the highly down-regulated group in PESCs compared to ESCs. Allele specific expression analysis of paternally-imprinted genes, i.e., those genes whose expression is down-regulated in PESCs, led to the identification of one novel candidate that was exclusively expressed from a paternal allele. Our findings suggest that PESCs could be used as a model for studying genomic imprinting and in the discovery of novel imprinted genes. Keywords: gene expression The transcriptomes of rhesus monkey embryonic stem cell lines derived from IVF-produced embryos (Oregon Rhesus Macaque Embryonic Stem, ORMES-22) were compared with rhesus monkey parthenogenetic embryonic stem cell lines (heterozygous rhesus Parthenogenetic embryonic stem cell lines, rPESC-2) and homozygous rhesus Parthenogenetic embryonic stem cell lines, ORMES-9). Moreover, the transcriptomes of rPESC-2 line were also compared with ORMES-9. Finally, the adult somatic skin fibroblasts were analyzed. Three biological replicates of each cell line (A, B, C) were analyzed.
Project description:Pluripotent stem cell lines derived from embryos of different stages have distinct pluripotent ground states, but similar levels of the transcription factor Oct4. Epiblast-derived pluripotent stem cells (EpiSCs), in contrast to embryonic stem (ES) cells, cannot form chimeras. We show that EpiSCs express lower levels of the transcription factors Sox2 and Klf4 than ES cells and have limited reprogramming potential, as shown by cell fusion. Sox2 overexpression dramatically increases the reprogramming potential, chimera formation, and germline contribution of EpiSCs. Therefore, although Oct4 is essential for reprogramming, the level of Sox2 defines both the reprogramming capability and the pluripotent ground states. RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates. 12 sample types were analyzed, each of them in duplicate. ESCm: Mouse ESC male; ESCf: Mouse ESC OG2 female; F9 EC: F9 EC (mouse embryonic carcinoma cell); F9-Sox2: F9 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCf: Mouse EpiSC OG2 female; Epi-Sox2f: Mouse EpiSC Sox2 (OG2 female) overexpressing wild type Sox2; P19 EC: P19 EC (mouse embryonic carcinoma cell); P19-Sox2: P19 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCm: Mouse EpiSC (GOF18 male) (duplicates); EpiSox2mL2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in condition EpiSC medium (CM); EpiSox2mE1: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like1); EpiSox2mE2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like2).