Mlana^tm1.2Bee

The targeting construct introduced loxP sites flanking the second and third exons and a frt-flanked neomycin resistance cassette (Pgk-neo) immediately upstream of the first loxP site, in the first intron. Mice bearing the resultant Mlanatm1Bee allele were crossed sequentially to FLP and Cre recombinase-expressing mice to delete the Pgk-neo cassette (producing Mlanatm1.1Bee) and the loxP-flanked exons, respectively. The deleted exons 2, containing the translation initiating ATG, and 3 together encode the transmembrane domain of the protein. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the absence of Mlana transcripts in skin of mice homozygous for the present mutation. No MLANA protein was detected either by western blot analysis of extracts of melanocytes cultured from the mutant mice or by immunohistologic analysis of these melanocytes or of skin sections using antibodies to the protein's C-terminus. (J:194886)