Chromosomal translocations induced at specified loci in human stem cells Journal Article

   


Authors: Brunet, E.; Simsek, D.; Tomishima, M.; DeKelver, R.; Choi, V. M.; Gregory, P.; Urnov, F.; Weinstock, D. M.; Jasin, M.
Article Title: Chromosomal translocations induced at specified loci in human stem cells
Abstract: The precise genetic manipulation of stem and precursor cells offers extraordinary potential for the analysis, prevention, and treatment of human malignancies. Chromosomal translocations are hallmarks of several tumor types where they are thought to have arisen in stem or precursor cells. Although approaches exist to study factors involved in translocation formation in mouse cells, approaches in human cells have been lacking, especially in relevant cell types. The technology of zinc finger nucleases (ZFNs) allows DNA double-strand breaks (DSBs) to be introduced into specified chromosomal loci. We harnessed this technology to induce chromosomal translocations in human cells by generating concurrent DSBs at 2 endogenous loci, the PPP1R12C/p84 gene on chromosome 19 and the IL2Rγ gene on the X chromosome. Translocation breakpoint junctions for t(19;X) were detected with nested quantitative PCR in a high throughput 96-well format using denaturation curves and DNA sequencing in a variety of human cell types, including embryonic stem (hES) cells and hES cell-derived mesenchymal precursor cells. Although readily detected, translocations were less frequent than repair of a single DSB by gene targeting or nonhomologous end-joining, neither of which leads to gross chromosomal rearrangements. While previous studies have relied on laborious genetic modification of cells and extensive growth in culture, the approach described in this report is readily applicable to primary human cells, including mutipotent and pluripotent cells, to uncover both the underlying mechanisms and phenotypic consequences of targeted translocations and other genomic rearrangements.
Keywords: unclassified drug; human cell; polymerase chain reaction; chromosome 19; dna damage; gene; gene targeting; in situ hybridization, fluorescence; dna repair; embryo; embryonic stem cell; cell line; gene locus; stem cell; gene rearrangement; dna breaks, double-stranded; stem cells; chromosome breakage; double-strand break repair (dsb repair); mesenchymal cells; nonhomologous end-joining (nhej); zinc finger nucleases; nuclease; zinc finger nuclease; chromosome rearrangement; chromosome translocation; chromosome translocation 19; double stranded dna break; il24gamma gene; mesenchymal stem cell; ppp1r12c gene; x chromosome; base sequence; chromosomes, human, pair 19; chromosomes, human, pair 6; chromosomes, human, x; dna breaks, single-stranded; embryonic stem cells; green fluorescent proteins; interleukin receptor common gamma subunit; translocation, genetic
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 106
Issue: 26
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 2009-06-30
Start Page: 10620
End Page: 10625
Language: English
DOI: 10.1073/pnas.0902076106
PUBMED: 19549848
PROVIDER: scopus
PMCID: PMC2700748
DOI/URL:

http://www.scopus.com/inward/record.url?eid=2-s2.0-67649757162&partnerID=40&md5=e2af2ff0cf99d3db7697562360d375f7
Notes: --- - "Cited By (since 1996): 9" - "Export Date: 30 November 2010" - "CODEN: PNASA" - "Source: Scopus"
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MSK Authors
  1. David Weinstock
    39 Weinstock
  2. Maria Jasin
    248 Jasin
  3. Mark J Tomishima
    45 Tomishima
  4. Erika Simone Suzanne Brunet
    6 Brunet
  5. Deniz Simsek
    5 Simsek
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