Project description:We showed the function of Rex1 in mouse ES cells as well as in embryos using the conventional gene targeting strategy. Our results clearly indicated that Rex1 function is dispensable for both the maintenance of pluripotency in ES cells and the development of embryos. However, Rex1-/- ES cells showed the defect to induce a subset of the marker genes of visceral endoderm, when differentiated as embryoid body, as found in EC cells. Conclusion: Rex1 should be regarded just as a marker of pluripotency without functional significance like the activity of alkaline phosphatase. Keywords: genetic modification design,reference design,replicate design
Project description:Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Differentiation of Rex1-GFPd2 ES cells was initiated by withdrawing 2i (Kalkan et al., 2016). Undifferentiated 2i-cells and post-2i withdrawal differentiating populations (16h, 25h-Rex1-High, 25h-Rex1-Low) were subjected to proteomic analysis by Mass Spectrometry.
Project description:To understand how pluripotency is controlled, we employed a modification of standard ChIP-chip method (biotin-mediated ChIP-chip) to identify targets of nine factors, including somatic cell reprogramming factors (Oct4, Sox2, Klf4, c-Myc) and others (Nanog, Dax1, Rex1, Zpf281, and Nac1) in mouse ES cells. We show the application of In vivo biotinylation mediated ChIP (bioChIP) to global target mapping. Our data also show that differential regulation of target genes correlates with the extent of promoter occupancy by multiple factors in maintaining stem cell pluripotency. Keywords: Biotin-mediated Chip-chip, Antibody ChIP-chip, Mouse embryonic stem cells
Project description:Mouse embryonic stem cells were maintained in minimal and chemically-defined culture conditions supporting naive pluripotency. Inhibitors of the Gsk3 (CHIR99021) and Mek/Erk (PD0325901) pathways were withdrawn, cultures maintained for 24 hours, and subsequently sorted by flow cytometry based on fluorescence of a short-half-life Rex1(Zfp42)::GFP reporter into two populations: Rex1-high cells, functionally capable of reversion to naive pluripotency, and Rex1-low cells that have exited the naive state.
Project description:Mouse embryonic stem cells were maintained in minimal and chemically-defined culture conditions supporting naive pluripotency. Inhibitors of the Gsk3 (CHIR99021) and Mek/Erk (PD0325901) pathways were withdrawn, cultures maintained for 24 hours, and subsequently sorted by flow cytometry based on fluorescence of a short-half-life Rex1(Zfp42)::GFP reporter into two populations: Rex1-high cells, functionally capable of reversion to naive pluripotency, and Rex1-low cells that have exited the naive state.
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a distinct formative phase of pluripotency preparatory to lineage priming
Project description:Zinc finger and SCAN domain-containing 10 (Zscan10, also known as Zfp206) encodes a transcription factor that has been reported to be involved in the maintenance of pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Zscan10 using the Cre-loxP system and analyzed its function. We succeeded in establishing Zscan10-null ES cells and confirmed their pluripotency by the generation of chimeric embryos. Our results clearly indicate that Zscan10 is dispensable for the ability of self-renewal and differentiation in ES cells.