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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.

AbstractMammalian somatic cells are usually diploid. Occasional rare human tumors have been shown to have a hypodiploid karyotype. We have isolated a near-haploid subclone (P1-55) from a heterogeneous human leukemia cell line, KBM-7. These near-haploid cells have approximately half the human diploid DNA content and have a haploid karyotype except for a disomy of chromosome 8 (25, XY, +8, Ph+). This cell line maintains a majority of cells with a near-haploid karyotype for at least 12 weeks in culture. By serial subcloning, we have isolated near-haploid subclones that maintain ploidy for at least 8 months in culture. Near-haploid cells can also be efficiently isolated from mixed ploidy cultures by size selection. The availability of this human near-haploid cell line should facilitate the genetic analysis of cultured human cells.

Topics: Karyotype (57%), Ploidy (56%), Somatic cell (53%), Cell culture (53%), Chromosome (52%)

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Citations
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Journal ArticleDOI
Abstract: CRISPR–Cas9 has been adopted as a powerful genome-editing technology in various species. By generating libraries of thousands of guide RNAs — which direct the Cas9 nuclease to chosen genomic loci — high-throughput genetic perturbations are now possible. This Review discusses the latest applications of CRISPR–Cas9 in mammalian functional genomics screens. It covers related genome-scale applications of Cas9 for either gene knockout or transcriptional modulation, and provides comparisons with complementary RNA interference (RNAi)-based approaches.

827 citations


Journal ArticleDOI
27 Nov 2009-Science
TL;DR: Using insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8, host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects ofdiphtheria toxin and exotoxin A are identified.
Abstract: Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.

452 citations


Journal ArticleDOI
24 Sep 2015-Cell
TL;DR: The consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3, which highlights fundamental principles of single-cell chromatin organization.
Abstract: Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.

330 citations


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

  • ...RESULTS Single-Cell DamID Methodology As a model system to develop single-cell DamID, we chose the humanmyeloid leukemia cell line KBM7, which is haploid for all chromosomes, except for chr8 and a small part of chr15 (Kotecki et al., 1999; Bürckstümmer et al., 2013)....

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  • ...To test this, we took advantage of the fact that KBM7 cells spontaneously form diploid cells at low frequency (Kotecki et al., 1999)....

    [...]

  • ...Single-Cell DamID Methodology As a model system to develop single-cell DamID, we chose the humanmyeloid leukemia cell line KBM7, which is haploid for all chromosomes, except for chr8 and a small part of chr15 (Kotecki et al., 1999; Bürckstümmer et al., 2013)....

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Journal ArticleDOI
06 Jun 2014-Science
TL;DR: Using a haploid genetic screen in human cells, it is found that ATP11C and CDC50A are required for aminophospholipid translocation from the outer to the inner plasma membrane leaflet; that is, they display flippase activity.
Abstract: Phospholipids are asymmetrically distributed in the plasma membrane. This asymmetrical distribution is disrupted during apoptosis, exposing phosphatidylserine (PtdSer) on the cell surface. Using a haploid genetic screen in human cells, we found that ATP11C (adenosine triphosphatase type 11C) and CDC50A (cell division cycle protein 50A) are required for aminophospholipid translocation from the outer to the inner plasma membrane leaflet; that is, they display flippase activity. ATP11C contained caspase recognition sites, and mutations at these sites generated caspase-resistant ATP11C without affecting its flippase activity. Cells expressing caspase-resistant ATP11C did not expose PtdSer during apoptosis and were not engulfed by macrophages, which suggests that inactivation of the flippase activity is required for apoptotic PtdSer exposure. CDC50A-deficient cells displayed PtdSer on their surface and were engulfed by macrophages, indicating that PtdSer is sufficient as an "eat me" signal.

324 citations


Journal ArticleDOI
TL;DR: ObLiGaRe is described, a new method for site-specific gene insertions that uses the efficient NHEJ pathway and acts independently of HR, and has enabled us to insert a 15-kb inducible gene expression cassette at a defined locus in human cell lines.
Abstract: Custom-designed nucleases (CDNs) greatly facilitate genetic engineering by generating a targeted DNA double-strand break (DSB) in the genome. Once a DSB is created, specific modifications can be introduced around the breakage site during its repair by two major DNA damage repair (DDR) mechanisms: the dominant but error-prone nonhomologous end joining (NHEJ) pathway, and the less-frequent but precise homologous recombination (HR) pathway. Here we describe ObLiGaRe, a new method for site-specific gene insertions that uses the efficient NHEJ pathway and acts independently of HR. This method is applicable with both zinc finger nucleases (ZFNs) and Tale nucleases (TALENs), and has enabled us to insert a 15-kb inducible gene expression cassette at a defined locus in human cell lines. In addition, our experiments have revealed the previously underestimated error-free nature of NHEJ and provided new tools to further characterize this pathway under physiological and pathological conditions.

277 citations


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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.
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