title: High throughput gene trapping and postinsertional modifications of gene trap alleles.
abstract: Gene trapping is a high-throughput insertional mutagenesis approach that has been primarily used in mouse embryonic stem cells (ESCs). As a high throughput technology, gene trapping helped to generate tenth of thousands of ESC lines harboring mutations in single genes that can be used for making knock-out mice. Ongoing international efforts operating under the umbrella of the International Knockout Mouse Consortium (IKMC; www.knockoutmouse.org) aim to generate conditional alleles for every protein coding gene in the mouse genome by high throughput conditional gene targeting and trapping. Here, we provide protocols for gene trapping in ESCs that can be easily adapted to any other mammalian cell. We further provide protocols for handling and verifying conditional gene trap alleles in ESC lines obtained from the IKMC repositories and describe a highly efficient method for the postinsertional modification of gene trap alleles. More specifically, we describe a protein tagging strategy based on recombinase mediated cassette exchange (RMCE) that enables protein localization and protein-protein interaction studies under physiological conditions.
authors.affiliation: Department for Molecular Hematology, University of Frankfurt Medical School, Theodor-Stern-Kai 7, Frankfurt am Main, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Schnütgen
authors.forename: Franziska
authors.initials: F
authors.lastname: Ehrmann
authors.forename: Patricia
authors.initials: P
authors.lastname: Ruiz-Noppinger
authors.forename: Harald
authors.initials: H
authors.lastname: von Melchner
mesh_terms: D000483:Alleles; D000818:Animals; D018929:Cell Culture Techniques; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology; D040461:Gene Components; D055786:Gene Knockout Techniques; D005822:Genetic Vectors; D051379:Mice; D018345:Mice, Knockout; D008979:Moloney murine leukemia virus / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D040901:Proteomics / Q000379:methods; D045522:Recombinases / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D014162:Transfection / Q000379:methods
chemical_list: D003470:Culture Media; D045522:Recombinases