Derivation of haploid embryonic stem cells from mouse embryos
TL;DR: Haploid mouse embryonic stem cells are described and their application in forward genetic screening is shown.
Abstract: Most animals are diploid, but haploid-only and male-haploid (such as honeybee and ant) species have been described. The diploid genomes of complex organisms limit genetic approaches in biomedical model species such as mice. To overcome this problem, experimental induction of haploidy has been used in fish. Haploid development in zebrafish has been applied for genetic screening. Recently, haploid pluripotent cell lines from medaka fish (Oryzias latipes) have also been established. In contrast, haploidy seems less compatible with development in mammals. Although haploid cells have been observed in egg cylinder stage parthenogenetic mouse embryos, most cells in surviving embryos become diploid. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening.
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TL;DR: The results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system and identify 27 known and 4 previously unknown genes implicated in these phenotypes.
Abstract: Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.
1,001 citations
TL;DR: It is shown that chromosomal compartments, topological-associated domains (TADs), contact insulation and long-range loops, all defined by bulk Hi-C maps, are governed by distinct cell-cycle dynamics, while loops are generally stable from G1 to S and G2 phase.
Abstract: Chromosomes in proliferating metazoan cells undergo marked structural metamorphoses every cell cycle, alternating between highly condensed mitotic structures that facilitate chromosome segregation, and decondensed interphase structures that accommodate transcription, gene silencing and DNA replication. Here we use single-cell Hi-C (high-resolution chromosome conformation capture) analysis to study chromosome conformations in thousands of individual cells, and discover a continuum of cis-interaction profiles that finely position individual cells along the cell cycle. We show that chromosomal compartments, topological-associated domains (TADs), contact insulation and long-range loops, all defined by bulk Hi-C maps, are governed by distinct cell-cycle dynamics. In particular, DNA replication correlates with a build-up of compartments and a reduction in TAD insulation, while loops are generally stable from G1 to S and G2 phase. Whole-genome three-dimensional structural models reveal a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data therefore allow re-interpretation of chromosome conformation maps through the prism of the cell cycle.
572 citations
TL;DR: In this paper, the authors employed a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state.
Abstract: Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.
229 citations
Cites background from "Derivation of haploid embryonic ste..."
...2009), mouse (Elling et al. 2011; Leeb and Wutz 2011) and rat (Li et al....
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Salk Institute for Biological Studies1, Gatsby Charitable Foundation2, University of California, San Diego3, Broad Institute4, McGovern Institute for Brain Research5, University of Pittsburgh6, National Institutes of Health7, University of Rochester8, Oregon Health & Science University9, Oregon National Primate Research Center10, Boston Children's Hospital11, Center for Neural Science12, RIKEN Brain Science Institute13, Keio University14, University of California, Los Angeles15, United States Department of Veterans Affairs16
TL;DR: Several recent advances have been described and their potential to advance the understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward are described.
221 citations
TL;DR: A genetic screen for silencing factors in X chromosome inactivation using haploid mouse embryonic stem cells that carry an engineered selectable reporter system identifies the RNA-binding protein Spen, the homolog of split ends, who is required for gene repression by Xist.
212 citations
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TL;DR: It is shown that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells and reveal that ES cells have an innate programme for self-replication that does not require extrinsics instruction.
Abstract: In the three decades since pluripotent mouse embryonic stem (ES) cells were first described they have been derived and maintained by using various empirical combinations of feeder cells, conditioned media, cytokines, growth factors, hormones, fetal calf serum, and serum extracts. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries, pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show, however, that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state.
3,250 citations
TL;DR: EZH1 partially complements Ezh2 in executing pluripotency during ESC differentiation, suggesting that cell-fate transitions require epigenetic specificity.
958 citations
TL;DR: Derivation of germline-competent ES cells from the rat paves the way to targeted genetic manipulation in this valuable biomedical model species and will also provide a refined test-bed for functional evaluation of pluripotent stem cell-derived tissue repair and regeneration.
798 citations
TL;DR: A full-length mouse Xist cDNA transgene and an inducible expression system facilitating controlled Xist expression in ES cells and differentiated cultures are generated, suggesting that reversible repression by Xist is a required initiation step that might occur during normal X inactivation in female cells.
586 citations
TL;DR: It is proposed that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells, and that in the absence of this signal, the entire ICM can acquire pluripotency.
Abstract: Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naive epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naive epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.
576 citations