Development of transposon-mediated GAL4gene and enhancer trap methods and production of abnormal behavioral phenotype by targeted expression of tetanus toxin light chain in zebrafish

Asakawa, K.1 , Mizusawa, K.1 , Urasaki, A.1 , Kotani, T.1 , Nagayoshi, S.1 , Kishimoto, Y.1 , Hibi, M.2 , Kawakami, K.1

1. National Institute of Genetics, 2. RIKEN CDB

The GAL4 trap system is a powerful method to modulate cellular functions in living animals by targeted gene expression. By using the Tol2 transposon system, we aimed to develop GAL4 gene and enhancer trap systems in zebrafish.
First, we created the Tol2 transposon constructs for GAL4 gene and enhancer trapping. A modified GAL4 gene was placed downstream of rabbit B-globin splice acceptor for gene trapping (SA-gal4) and the zebrafish hsp70 promoter for enhancer trapping (hsp-gal4). Then, we created random insertions of these Tol2 constructs in the zebrafish genome. By crossing the SA-gal4 fish or the hsp-gal4 fish with the UAS-EGFP reporter fish, we isolated 206 GAL4 lines based on unique GFP expression patterns. The expression pattern of the GAL4 mRNA was consistent with the GFP expression, indicating that these GAL4 trap constructs are regulated by endogenous promoters or locus-specific enhancers.
To test if these GAL4 lines can be used to modify neuronal functions, we constructed the transgenic fish carrying a gene for tetanus toxin light chain (TeTxLC), which blocks neurotransmitter release via synaptic vesicles, downstream of UAS. By crossing the UAS-TeTxLC line with each of 119 GAL4 lines, we identified 9 GAL4 lines that showed a touch response defect. We think that in such lines the Gal4 was expressed in neurons that are involved in the touch response and synaptic transmission of such neurons was blocked by induction of TeTxLC.