The long term objective of this work is to develop a new method for rapidly disrupting auditory gene function in vivo. Our specific aims are to build a vector that is optimal for both efficient shRNA knockdown and activation of a Venus reporter, and then to validate that this vector effectively inactivates gene function in the auditory system in vivo. To achieve these aims, we are testing a variety of shRNA vectors by introducing them to embryonic stem cells, using knock-down of the stem cell differentiation factor Oct4 as a read-out. Once we have identified the best vector, i.e. that which phenocopies loss of Oct4 in ES cells, then we will build a vector carrying shRNA against the Gata3 gene, introduce the vector to ES cells, and use these cells to create conditional Gata3 shRNA mice. These mice will be crossed to a variety of Cre lines to test whether shRNA-mediated knockdown reproduces the known effects of mutations in Gata3.