Global gene disruption in human cells to assign genes to phenotypes
Name
Ploegh_Global gene.pdf
Size
1.51 MB
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Adobe PDF
Checksum (MD5)
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Author(s)
Ploegh, Hidde
Yuan, Bingbing B.
Carette, Jan E.
Guimaraes, Carla P.
Wuethrich, Irene
Blomen, Vincent A.
Varadarajan, Malini
Sun, Chong
Bell, George W.
Muellner, Markus K.
Date Issued
May 2011
Journal
Nature Biotechnology
Publisher
Nature Publishing Group
Citation
Carette, Jan E et al. “Global Gene Disruption in Human Cells to Assign Genes to Phenotypes by Deep Sequencing.” Nature Biotechnology 29.6 (2011): 542–546.
Version
Author's final manuscript
Abstract
Insertional mutagenesis in a haploid background can disrupt gene function[superscript 1]. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones[superscript 1] enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites[superscript 2]. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Whitehead Institute for Biomedical Research
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike 3.0
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DOI of Published Version
https://doi.org/10.1038/nbt.1857
