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 β-galactosidase reporter are given.
Key wordsGene trap Gene disruption Promoter activity Embryonic stem cells Mouse genome β-Galactosidase β-Geo X-gal staining
The authors would like to thank Tobias Fischer and Peter M. Benz for providing organs of mice with different genes trapped to demonstrate X-Gal stainings, and Karin Bundschu for providing X-Gal stained tissue sections of mice with a trapped spred2 gene.