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Journal ArticleDOI

Isolation and Characterization of a Near-Haploid Human Cell Line

01 Nov 1999-Experimental Cell Research (Academic Press)-Vol. 252, Iss: 2, pp 273-280
TL;DR: By serial subcloning, a near-haploid subclone (P1-55) is isolated from a heterogeneous human leukemia cell line, KBM-7, which has approximately half the human diploid DNA content and has a haploid karyotype except for a disomy of chromosome 8.
About: This article is published in Experimental Cell Research.The article was published on 1999-11-01. It has received 136 citations till now. The article focuses on the topics: Karyotype & Ploidy.
Citations
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Journal ArticleDOI
TL;DR: How PtdSer is exposed in apoptotic cells and in activated platelets is described and discussed, and Ptd Ser exposure in other biological processes is discussed.
Abstract: Phosphatidylserine (PtdSer) is a phospholipid that is abundant in eukaryotic plasma membranes. An ATP-dependent enzyme called flippase normally keeps PtdSer inside the cell, but PtdSer is exposed by the action of scramblase on the cell's surface in biological processes such as apoptosis and platelet activation. Once exposed to the cell surface, PtdSer acts as an 'eat me' signal on dead cells, and creates a scaffold for blood-clotting factors on activated platelets. The molecular identities of the flippase and scramblase that work at plasma membranes have long eluded researchers. Indeed, their identity as well as the mechanism of the PtdSer exposure to the cell surface has only recently been revealed. Here, we describe how PtdSer is exposed in apoptotic cells and in activated platelets, and discuss PtdSer exposure in other biological processes.

291 citations


Cites methods from "Isolation and Characterization of a..."

  • ...To identify plasma-membrane PtdSer flippases in mammalian cells, we performed a forward genetic screen using KBM7,(22) a human myeloid-cell line with a near-haploid karyotype.(23) KBM7 cells were randomly mutagenized with gene-trap retroviruses,(24) and cells that could not efficiently incorporate fluorescently labeled PtdSer were collected by repeated cell sorting....

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Journal ArticleDOI
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 "Isolation and Characterization of a..."

  • ...loid leukemia patient and stably cultured over several months (Kotecki et al. 1999)....

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Journal ArticleDOI
03 Nov 2011-Nature
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.

228 citations


Cites background from "Isolation and Characterization of a..."

  • ...20 for a review), and a near-haploid human-tumour-derived cell line has been describe...

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Journal ArticleDOI
20 May 2010-Blood
TL;DR: The generation and characterization of iPSCs derived from human chronic myeloid leukemia cells are described and it is shown that, despite the presence of oncogenic mutations, these cells acquired pluripotency by the expression of 4 transcription factors and underwent differentiation into cell types derived of all 3 germ layers during teratoma formation.

221 citations

09 Apr 2015
TL;DR: Recent advances using Cas9 for genome-scale screens are described, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity.
Abstract: Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges.

217 citations

References
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Journal ArticleDOI
16 Jun 1989-Science
TL;DR: The current status of gene targeting with particular emphasis on germ line modification of the mouse genome is discussed, and the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred are described.
Abstract: Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.

2,320 citations

Journal ArticleDOI
28 May 1981-Nature
TL;DR: Clones of homozygous fish have been produced from individual homozygotes and associated genetic methods facilitate genetic analyses of this vertebrate.
Abstract: Homozygous diploid zebra fish have been produced on a large scale by the application of simple physical treatments. Clones of homozygous fish have been produced from individual homozygotes. These clones and associated genetic methods will facilitate genetic analyses of this vertebrate.

1,203 citations

Journal ArticleDOI
08 Aug 1997-Science
TL;DR: At the checkpoint between the prereplicative phase of growth and the phase of chromosome replication, cells lacking p21 failed to arrest the cell cycle in response to DNA damage, but their apoptotic response and genomic stability were unaltered.
Abstract: Most somatic cells die after a finite number of cell divisions, a phenomenon described as senescence. The p21 CIP1/WAF1 gene encodes an inhibitor of cyclin-dependent kinases. Inactivation of p21 by two sequential rounds of targeted homologous recombination was sufficient to bypass senescence in normal diploid human fibroblasts. At the checkpoint between the prereplicative phase of growth and the phase of chromosome replication, cells lacking p21 failed to arrest the cell cycle in response to DNA damage, but their apoptotic response and genomic stability were unaltered. These results establish the feasibility of using gene targeting for genetic studies of normal human cells.

827 citations

Journal Article
TL;DR: The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.
Abstract: Rat fibroblast cell lines with targeted disruptions of both c-myc gene copies were constructed. Although c-myc null cells are viable, their growth is significantly impaired. The absence of detectable N-myc or L-myc expression indicates that Myc function is not absolutely essential for cell viability. The c-myc null phenotype is stable and can be reverted by introduction of a c-myc transgene. Exponentially growing c-myc null cells have the same cell size, rRNA, and total protein content as their c-myc +1+ parents, but the rates of RNA and protein accumulation as well as protein degradation are reduced. Both the G1 and G2 phases of the cell cycle are significantly lengthened, whereas the duration of S phase is unaffected. This is the first direct demonstration of a requirement for c-myc in G2. The G0-+S transition is synchronous, but S-phase entry is significantly delayed. The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.

473 citations

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