This proposal is a technology-driven pilot project to develop techniques to disrupt genes specifically in the mouse inner ear. The mouse has been used successfully as a model system to understand disorders of hearing and balance, and the ability to disrupt (or """"""""knock out"""""""") genes in mice has proved of great use in understanding gene function in the inner ear. However, studies of gene function in the inner ear using gene knockout technology are often hampered by premature lethality, or by indirect effects of gene deletion in other tissues. The ability to analyze the ear phenotype of mutant mice at all stages of development including the mature adult would be of enormous benefit to the hearing and balance research community. This proposal makes use of recently developed """"""""Cre-Lox"""""""" technology, in which a gene flanked by LoxP DNA elements can be excised, and hence inactivated, by the Cre recombinase protein. Mating mice in which a gene of interest is flanked by LoxP sites with mice that express the Cre recombinase only in the inner ear, will excise the gene specifically in the inner ear and nowhere else. In order to develop this technology, we require a promoter that will drive gene expression only in the inner ear. We have obtained a 2.7kb promoter fragment of the ADH-4 alcohol dehydrogenase gene that directs expression in the primordium of the inner ear - the otic placode - as well as the hindbrain and neural crest.
The aim of this project is to isolate fragments of the ADH-4 promoter that drive gene expression only in the otic placode and to use these fragments to construct transgenic mouse lines in which the promoter fragments drive expression of the Cre recombinase gene only in the otic placode. Many mouse lines now exist in which different genes have been flanked (""""""""floxed"""""""") by LoxP sites. In the future, we and others will be able to mate the Cre expressing mice produced in this pilot study with such """"""""floxed"""""""" mouse lines to disrupt genes specifically in the inner ear and to analyze the effect of gene disruption at all stages of mouse development.
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