A floxed neo cassette and a single loxP site flanking exons 2 and 3 were introduced via homologous recombination. The floxed neo cassette and exons 2 and 3 were removed by transient expression of cre recombinase in correctly targeted cells, resulting in deletion of sequences encoding 5 of the 6 transmembrane segments of the Hcn2 channel as well as part of the pore. Absence of gene expression was confirmed by Northern and Western blot analysis of brain extracts from homozygous mutant animals.