Ex Vivo Transposon-Mediated Genetic Screens for Cancer Gene Discovery

Kathryn A O'Donnell-Mendell, Yabin Guo, Shruthy Suresh, Barrett L. Updegraff, Xiaorong Zhou

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Transposon mutagenesis has emerged as a powerful methodology for functionally annotating cancer genomes. Although in vivo transposon-mediated forward genetic screens have proven to be valuable for cancer gene identification, they are also time consuming and resource intensive. To facilitate the rapid and cost-effective identification of genes that regulate tumor-promoting pathways, we developed a complementary ex vivo transposon mutagenesis approach wherein human or mouse cells growing in culture are mutagenized and screened for the acquisition of specific phenotypes in vitro or in vivo, such as growth factor independence or tumor-forming ability. This approach allows discovery of both gain- and loss-of-function mutations in the same screen. Transposon insertions sites are recovered by high-throughput sequencing. We recently applied this system to comprehensively identify and validate genes that promote growth factor independence and transformation of murine Ba/F3 cells. Here we describe a method for performing ex vivo Sleeping Beauty-mediated mutagenesis screens in these cells, which may be adapted for the acquisition of many different phenotypes in distinct cell types.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages145-157
Number of pages13
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1907
ISSN (Print)1064-3745

Keywords

  • Cell-based screens
  • DNA transposons
  • Ex vivo forward genetic screening
  • Functional annotation
  • Growth-factor independence
  • Sleeping Beauty
  • Transposon mutagenesis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    O'Donnell-Mendell, K. A., Guo, Y., Suresh, S., Updegraff, B. L., & Zhou, X. (2019). Ex Vivo Transposon-Mediated Genetic Screens for Cancer Gene Discovery. In Methods in Molecular Biology (pp. 145-157). (Methods in Molecular Biology; Vol. 1907). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8967-6_12