Cnr1^tm1.1Kpm

An HA epitope sequence, two mutations that change serines 426 and 430 to alanines (S426A, S430A), and a neomycin phosphotransferase (NeoR) gene flanked by frt sequences were inserted via homologous recombination. Serines 426 and 430 are putative GRK phosphorylation sites that when mutated to alanines are nonphosphorylatable and result in a desensitization resistant form of Cnr1. The frt-flanked NeoR gene was removed via flp-mediated recombination. Southern blot confirmed expression of a 4.2 kb mutant fragment and PCR confirmed expression of the mutant mRNA. Western blot analysis confirmed that protein levels in the brain remain unchanged except for a 15% decrease in the cerebellum. (J:208116)