Transgenic medaka fish which mimic the endogenous expression of neuronal kinesin, KIF5A.

ABSTRACT

Intracellular transport is spatiotemporally controlled by microtubule-dependent motor proteins, including kinesins. In order to elucidate the mechanisms controlling kinesin expression, it is important to analyze their genomic regulatory regions. In this study, we cloned the neuronal tissue-specific kinesin in medaka fish and generated transgenic fish which mimic endogenous neuronal kinesin expression in order to elucidate the mechanisms which regulate kinesin expression. Searches for medaka neuronal orthologues by RT-PCR identified a candidate gene expressed only in neuronal tissues. Using BAC clones, we determined the cDNA sequence and the gene structure of the candidate neuronal kinesin. Evolutionary analysis indicated that the candidate gene encoded medaka KIF5Aa. The endogenous medaka orthologue was found to be expressed only in the nervous system, including the brain and spinal cord, while expression of KIF5Ab was not exclusive to neuronal tissues. Transgenic (Tg) medaka that expressed EGFP under the control of the 6.9 kbp 5' and 1.9kbp 3' flanking regions of the KIF5Aa gene showed characteristic expression throughout the nervous system, including the brain, spinal cord, olfactory pit, eye and cranial nerve. Immunohistological analysis showed that EGFP expression in Tg fish co-localized with expression of HuC/D, a neuronal marker. These results demonstrate that the 6.9 kbp 5' and 1.9 kbp 3' flanking regions of medaka KIF5Aa have neuronal-specific promoter activity mimicking endogenous expression of medaka KIF5Ab. This transgenic fish strain will be useful for further functional analysis of the effects of these regulatory regions on gene expression.