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During evolution, the mammalian middle ear apparatus evolved from jawbones in non-mammalian vertebrates. The nkx3.2 gene has shown expression in the jaw joint region, while in mammalian vertebrates expression was active in the middle ear (Wilson and Tucker, 2004; Newman, et al., 1997; Tucker, et al., 2004). The conservation of genes is a key question in evolutionary biology and while these findings support the idea that nkx3.2 is a conserved gene, hardly anything is known about the corresponding enhancer. The aim of this project is to create a transgenic line of zebrafish with the nkx3.2 enhancer sequence from zebrafish and other species containing the fluorescent protein mCherry to see if the putative nkx3.2 enhancer sequences found in mammalian and non-mammalian are evolutionarily conserved between the different species. The first step of this project is to create the necessary expression clones. Deoxyribonucleic acid (DNA) isolation, primer design and Polymerase Chain Reaction (PCR) are first executed. The resulting PCR products were the putative enhancer sequences of Danio rerio (Zebrafish), Homo sapiens (Human), Mus musculus (House mouse), Xenopus tropicalis (Western clawed frog), Polypterus bichir (Nile bichir) and Callorhinchus milii (Elephant shark). The expression clones are constructed using the Gateway cloning technique. This is a two-step process: cloning the gene of interest into a donor vector using the BP reaction; and subcloning the gene of interest from the Entry clone into a Destination vector using the LR reaction that produces the Expression clone. Secondly a transposon-donor plasmid and synthetic mRNA encoding the transposase are introduced into fertilized eggs by microinjection. The transposase protein is translated from the injected mRNA and catalyzes excision of the transposon construct from the donor plasmid, leading to stable integration of the excised DNA in the genome. The integration occurs during early stages of embryonic develo