Gene trap: knockout on the fast lane.
Gene trapping is a powerful tool to ablate gene function and to analyze in vivo promoter activity of the trapped gene in parallel. The gene trap strategy is not as commonly used as the conventional gene-targeting strategy, although it offers appealing options. Nowadays, a wide collection of embryonic stem cell clones, with a huge variety of trapped genes, have been identified and are available through the members of the International Gene Trap Consortium (IGTC). This chapter focuses on BLAST searches for the appropriate stem cell clones, the confirmation of vector insertion by RT-PCR or X-Gal staining, and the characterization of the exact insertion site to develop a PCR-based genotyping strategy. Furthermore, protocols to follow the activity of the commonly used beta-galactosidase reporter are given.
Ullrich
Melanie
M
Institute of Physiology I, University of Wuerzburg, Wuerzburg, Germany.
Schuh
Kai
K
D000818:Animals; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology; D053595:Embryonic Stem Cells / Q000502:physiology; D005822:Genetic Vectors; D051379:Mice / Q000196:embryology / Q000235:genetics*; D018345:Mice, Knockout; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*