title: piggyBac transposon-based insertional mutagenesis in mouse haploid embryonic stem cells.
abstract: Forward genetic screening is a powerful non-hypothesis-driven approach to unveil the molecular mechanisms and pathways underlying phenotypes of interest. In this approach, a genome-wide mutant library is first generated and then screened for a phenotype of interest. Subsequently, genes responsible for the phenotype are identified. There have been a number of successful screens in yeasts, Caenorhabditis elegans and Drosophila. These model organisms all allow loss-of-function mutants to be generated easily on a genome-wide scale: yeasts have a haploid stage in their reproductive cycles and the latter two organisms have short generation times, allowing mutations to be systematically bred to homozygosity. However, in mammals, the diploid genome and long generation time have always hampered rapid and efficient production of homozygous mutant cells and animals. The recent discovery of several haploid mammalian cell lines promises to revolutionize recessive genetic screens in mammalian cells. In this protocol, we describe an overview of insertional mutagenesis, focusing on DNA transposons, and provide a method for an efficient generation of genome-wide mutant libraries using mouse haploid embryonic stem cells.
authors.affiliation: Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, UK.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Pettitt
authors.forename: E-Pien
authors.initials: EP
authors.lastname: Tan
authors.forename: Kosuke
authors.initials: K
authors.lastname: Yusa
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D004251:DNA Transposable Elements; D053595:Embryonic Stem Cells / Q000378:metabolism*; D006238:Haploidy; D007150:Immunohistochemistry; D051379:Mice; D016254:Mutagenesis, Insertional
chemical_list: D004251:DNA Transposable Elements