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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: This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.
Abstract: The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

37 citations


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

  • ...KBM7 cells were originally isolated from a patient with chronic myeloid leukaemia [18] and are haploid apart from disomy of chromosome 8 and the sex chromosomes [19]....

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Journal ArticleDOI
TL;DR: These comparative haploid genetic screens demonstrate that, despite their similarity in overall architecture and subcellular localization, GPI-APs follow markedly distinct biosynthetic and trafficking pathways.

35 citations


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

  • ...Recently, multiple mammalian haploid cell lines, including tumor cells and pluripotent stem cells, were isolated (Kotecki et al., 1999; Carette et al., 2011b; Yang et al., 2012; Li et al., 2012; Leeb and Wutz, 2011)....

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Journal ArticleDOI
TL;DR: Haploid ESCs are characterized by a wide developmental potential and can contribute to chimeric embryos and mice and could offer an unprecedented tool for genome exploration in the future.
Abstract: Most animal genomes are diploid, and mammalian development depends on specific adaptations that have evolved secondary to diploidy. Genomic imprinting and dosage compensation restrict haploid development to early embryos. Recently, haploid mammalian development has been reinvestigated since the establishment of haploid embryonic stem cells (ESCs) from mouse embryos. Haploid cells possess one copy of each gene, facilitating the generation of loss-of-function mutations in a single step. Recessive mutations can then be assessed in forward genetic screens. Applications of haploid mammalian cell systems in screens have been illustrated in several recent publications. Haploid ESCs are characterized by a wide developmental potential and can contribute to chimeric embryos and mice. Different strategies for introducing genetic modifications from haploid ESCs into the mouse germline have been further developed. Haploid ESCs therefore introduce new possibilities in mammalian genetics and could offer an unprecedented...

34 citations

Journal ArticleDOI
Nadia Martinez-Martin1
TL;DR: This review discusses available technologies for the high throughput study of extracellular protein interactions between pathogens and their hosts, with a focus on mammalian viruses and bacteria.
Abstract: Pathogens have evolved unique mechanisms to breach the cell surface barrier and manipulate the host immune response to establish a productive infection. Proteins exposed to the extracellular environment, both cell surface-expressed receptors and secreted proteins, are essential targets for initial invasion and play key roles in pathogen recognition and subsequent immunoregulatory processes. The identification of the host and pathogen extracellular molecules and their interaction networks is fundamental to understanding tissue tropism and pathogenesis and to inform the development of therapeutic strategies. Nevertheless, the characterization of the proteins that function in the host-pathogen interface has been challenging, largely due to the technical challenges associated with detection of extracellular protein interactions. This review discusses available technologies for the high throughput study of extracellular protein interactions between pathogens and their hosts, with a focus on mammalian viruses and bacteria. Emerging work illustrates a rich landscape for extracellular host-pathogen interaction and points towards the evolution of multifunctional pathogen-encoded proteins. Further development and application of technologies for genome-wide identification of extracellular protein interactions will be important in deciphering functional host-pathogen interaction networks, laying the foundation for development of novel therapeutics.

32 citations


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

  • ...In initial studies, Carette and colleagues took advantage of the KBM7 cell line, a derivative of the chronic myeloid leukemia cell line (CML) with a haploid karyotype except for chromosome 8 [130]....

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Journal ArticleDOI
08 Jul 2013-PLOS ONE
TL;DR: The first reporter screen in the near-haploid KBM7 cell line is performed to identify constitutive inhibitors of NF-κB, demonstrating that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors.
Abstract: The development of forward genetic screens in human haploid cells has the potential to transform our understanding of the genetic basis of cellular processes unique to man. So far, this approach has been limited mostly to the identification of genes that mediate cell death in response to a lethal agent, likely due to the ease with which this phenotype can be observed. Here, we perform the first reporter screen in the near-haploid KBM7 cell line to identify constitutive inhibitors of NF-κB. CYLD was the only currently known negative regulator of NF-κB to be identified, thus uniquely distinguishing this gene. Also identified were three genes with no previous known connection to NF-κB. Our results demonstrate that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors.

32 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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