| The Gal4/UAS transcriptional activation system of yeast has been used extensively to manipulate gene expression in Drosophila. Although its use has been more limited in zebrafish, with the discovery of the Tol2 transposition system for efficient transgenesis, Gal4/UAS utilization has expanded greatly in recent years. We performed a Tol2-mediated gene/enhancer trap screen (Davison et al., 2007), which resulted in several transgenic zebrafish lines with a variety of tissue-restricted Gal4 expression patterns. These lines are useful reagents that can be used for tissue-specific expression other UAS-regulated transgenes.;Although expression from UAS-regulated transgenes is initially robust, it becomes variegated and silenced over progressive generations. Further investigation revealed that transgenes of silenced larvae are methylated at the multi-copy 14X UAS (Goll et al., 2009). We have attempted to minimize methylation-induced transgene silencing by reducing the number of tandem repeats in the UAS site. Using four distinct UAS sequences, we created a less repetitive 4X UAS that is capable of achieving robust expression levels (Akitake et al., 2011). Transgenerational analysis of stable transgenic lines over three generations revealed that the 4X UAS is less methylated and silenced than the original 14X UAS.;We aim to use the optimized Gal4/UAS system to ablate defined brain regions and test their behavioral consequences. To generate new Gal4 driver lines with habenular expression, we tested upstream sequence from two genes for their ability to direct transgene expression in the zebrafish larval habenulae. Using a modified BAC recombineering approach, we successfully employed Tol2-mediated BAC transgenesis to create a habenula-specific Gal4 driver line. The transgenic line can be used to for tissue-restricted expression of UAS-regulated transgenes and will facilitate the selective ablation of the larval habenulae. |
