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Articles by I. M Cheeseman
Total Records ( 2 ) for I. M Cheeseman
  R. C DeKelver , V. M Choi , E. A Moehle , D. E Paschon , D Hockemeyer , S. H Meijsing , Y Sancak , X Cui , E. J Steine , J. C Miller , P Tam , V. V Bartsevich , X Meng , I Rupniewski , S. M Gopalan , H. C Sun , K. J Pitz , J. M Rock , L Zhang , G. D Davis , E. J Rebar , I. M Cheeseman , K. R Yamamoto , D. M Sabatini , R Jaenisch , P. D Gregory and F. D. Urnov

Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.

  M Amano , A Suzuki , T Hori , C Backer , K Okawa , I. M Cheeseman and T. Fukagawa

The constitutive centromere-associated network (CCAN) proteins are central to kinetochore assembly. To define the molecular architecture of this critical kinetochore network, we sought to determine the full complement of CCAN components and to define their relationships. This work identified a centromere protein S (CENP-S)–containing subcomplex that includes the new constitutive kinetochore protein CENP-X. Both CENP-S– and CENP-X–deficient chicken DT40 cells are viable but show abnormal mitotic behavior based on live cell analysis. Human HeLa cells depleted for CENP-X also showed mitotic errors. The kinetochore localization of CENP-S and -X is abolished in CENP-T– or CENP-K–deficient cells, but reciprocal experiments using CENP-S–deficient cells did not reveal defects in the localization of CCAN components. However, CENP-S– and CENP-X–deficient cells show a significant reduction in the size of the kinetochore outer plate. In addition, we found that intrakinetochore distance was increased in CENP-S– and CENP-X–deficient cells. These results suggest that the CENP-S complex is essential for the stable assembly of the outer kinetochore.

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