Real-time masurement of circadian gene expresion in peripheral tissues from transgenic zebrafish

KANEKO M; HERNÁNDEZ DE BORSETTI N; CAHILL G

SICILY

Conferencia; FOURTH STROMBOLI CONFERENCE ON AGING AND CANCER STROMBOLI AEOLIAN ISLANDS SICILY ITALY; 2005

Valteri Pierpaoli Institutum Studiis Naturae ac humanae Vitae Provehendis

In vivo recordings of bioluminescence rhythms have been used in several organisms suchas cyanobacteria, plants, Drosophila, and mammals with great success in mutagenesisscreening as well as studies on physiological aspects of circadian rhythms. In this regard,zebrafish is the most convenient vertebrate species for large-scale mutagenesis. To developa method for monitoring circadian gene expression in vivo, we have generated transgeniczebrafish in which cyclical expression of firefly luciferase (luc) gene is driven by a clockgenepromoter. We first constructed a transgene in which luc is fused to the promoter ofzebrafish Per3 gene whose expression is known to cycle in a circadian manner. Three of fivegermline transformant lines were found to show bioluminescence rhythms in larvae. Thislarval rhythm was shown to reflect mRNA cycling of endogenous zPer3, and peaks duringthe subjective day. The peak phase was shifted earlier in a short-period mutant g4 comparedto wild type. Therefore, this system may be used to screen for new circadianmutants. In adults, many peripheral tissues such as the retina, heart, and spleen showedrobust oscillations of bioluminescence in culture. These rhythms in cultured tissues wereobserved in light:dark cycles as well as in constant conditions, and can be entrained bylight:dark cycles. The bioluminescence rhythms peak in the middle of the subjective day inseveral different tissues. These lines should be useful for identification of new circadianclock mutations.