Targeted Gene Expression by Tol2 Transposon-mediated Gal4 Gene and Enhancer Trap Approaches in Zebrafish

Kazuhide Asakawa1, Maximiliano Suster1, Kanta Mizusawa1, Saori Nagayoshi1, Tomoya Kotani1, Akihiro Urasaki1, Yasuyuki Kishimoto1, Masahiko Hibi2, and Koichi Kawakami1,3

1) National Institute of Genetics, Mishima, Japan, 2) RIKEN CDB,Kobe, Japan , 3)The Graduate University for Advanced Studies (SOKENDAI), Japan

  We report here the development of a novel targeted gene expression methodology in zebrafish. First, we developed an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS). We showed that Gal4FF can activate transcription through UAS in zebrafish. To collect fish that express Gal4FF in spatially and temporally restricted patterns, we have been performing a large scale Gal4FF gene trap and enhancer trap screens by using the Tol2 transposon system. To date, we generated more than 300 trap lines that expressed Gal4FF in specific tissues, cells, and organs, demonstrating that these Gal4FF trap constructs can effectively isolate transgenic Gal4FF lines. To demonstrate that specific cellular function can be modified by the Gal4FF-UAS system, we developed a transgenic effector fish carrying the tetanus toxin light chain (TeTxLC:CFP) gene downstream of UAS, which blocks synaptic transmission. By crossing the UAS:TeTxLC fish with Gal4FF transgenic fish, we identified more than ten lines that exhibited defects in embryonic behaviors. We found that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF-UAS system and Gal4FF gene trap and enhancer trap methods should provide an important targeted gene expression methodology in zebrafish. In the course of these studies, we found that (1) the cis sequences in the trap constructs caused background expression of Gal4FF and that (2) the UAS transgenes were susceptible to chromosomal position effect. We will discuss what can improve targeted gene expression by the Gal4UAS system in zebrafish.