Protocol

In Vitro Mutagenesis Protocols

Volume 634 of the series Methods in Molecular Biology pp 331-342

Date:

Phenotype Based Functional Gene Screening Using Retrovirus-Mediated Gene Trapping in Quasi-Haploid RAW 264.7 Cells

  • Sung Ouk KimAffiliated withInfectious Diseases Research Group, Department of Microbiology and Immunology, Siebens-Drake Research Institute, University of Western Ontario Email author 
  • , Soon-Duck HaAffiliated withInfectious Diseases Research Group, Department of Microbiology and Immunology, Siebens-Drake Research Institute, University of Western Ontario

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Abstract

In vitro random mutagenesis, followed by phenotype screening, provides a rapid and convenient tool for identifying novel genes involved in the phenotype of interest. However, the forward mutagenic approach in mammalian somatic cells is seriously limited by the diploidic nature of the genome. To overcome this impediment, we developed a method that allows functional screening for both haploid insufficient and sufficient genes involved in the phenotype of interest, utilizing a retrovirus gene trap mutagenesis in chemical mutagen-generated quasi-haploid cells. This method was used to identify novel host genes that are required for macrophage sensitivity to anthrax lethal toxin.

Key words

Retrovirus Gene-trapping Forward genetics Macrophages Cell death