ABSTRACT
Systems ............................................................................................. 173 8.2.3 Detection of the Introduced Mutations Using PCR and Southern
Blotting ............................................................................................. 176 8.2.4 Knock-In of Reporter Genes ............................................................. 176 8.2.5 Knock-Add-On Mouse Models ......................................................... 177 8.2.6 Generation of Genetically Modi—ed Mouse Models Using Gene
Trapping ............................................................................................ 178 8.3 Protocols for Gene Targeting Experiments .................................................. 179
8.3.1 Mouse Embryonic Fibroblasts (MEFs) ............................................. 179 8.3.1.1 Preparation of MEFs .......................................................... 179 8.3.1.2 Harvesting EF Cells and γ-Irradiation ............................... 180 8.3.1.3 Thawing and Replating EF Cells ....................................... 181
8.3.2 Mouse Embryonic Stem Cells .......................................................... 181 8.3.2.1 ES Cells-General Aspects of Handling and Use ............ 181 8.3.2.2 Passage of ES Cells ............................................................ 183
8.3.3 Electroporation ................................................................................. 184 8.3.4 Picking ES Cell Clones ..................................................................... 185 8.3.5 Freezing ES Cell Clones ................................................................... 186 8.3.6 Preparation of Genomic DNA from ES Cells .................................. 188
Acknowledgment ................................................................................................... 189 References .............................................................................................................. 189
Transient receptor potential (TRP) proteins constitute a superfamily of cationpermeable channels that show a broad range of activation, regulation, and selectivity mechanisms.1,2 Four TRP proteins are assumed to form homo-oligomeric and heterooligomeric channels, and the biological roles of TRP channels appear to be highly diverse. They essentially contribute to thermosensation and pain perception, Ca2+
and Mg2+ absorption, endothelial permeability, smooth muscle proliferation, and mast cell degranulation in mice and other mammals.3-5 The majority of our knowledge of the properties of TRP channels arises from diseases linked to mutations in TRP channels6,7 and from recordings of the activity of ectopically expressed TRP channel proteins in cell lines such as HEK 293 cells. In this environment, they do not necessarily act in accordance with the native cellular context as in primary cells.
