In Vitro Mutagenesis Protocols pp 331-342

Part of the Methods in Molecular Biology book series (MIMB, volume 634)

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

Protocol

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 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Infectious Diseases Research Group, Department of Microbiology and Immunology, Siebens-Drake Research InstituteUniversity of Western OntarioLondonCanada

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