A 1.6-kb fragment containing exons 6-8 was replaced with a nuclear localization signal and LacZ reporter gene, the Engrailed-2 intron and splice acceptor site (En-2/SA), and an internal ribosome entry site followed by a loxP-flanked phosphoglycerine kinase (PGK)-neomycin resistance gene. The loxP-flanked PGK-neomycin resistance gene was removed via Cre-mediated recombination in the germline. Western blot analysis using a polyclonal anti-TMC6 antibody confirmed the complete absence of protein expression in cell lysates from homozygous mutant thymocytes.