RRC ID 4116
Author Asakawa K, Suster ML, Mizusawa K, Nagayoshi S, Kotani T, Urasaki A, Kishimoto Y, Hibi M, Kawakami K.
Title Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.
Journal Proc. Natl. Acad. Sci. U.S.A.
Abstract Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying 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) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered 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 gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.
Volume 105(4)
Pages 1255-60
Published 2008-1-29
DOI 10.1073/pnas.0704963105
PII 0704963105
PMID 18202183
PMC PMC2234125
MeSH 5' Untranslated Regions / genetics Animals Animals, Genetically Modified Crosses, Genetic DNA Transposable Elements / genetics DNA Transposable Elements / physiology* DNA-Binding Proteins Enhancer Elements, Genetic / genetics* Gene Expression Regulation, Developmental / genetics Gene Targeting* Genes, Reporter Green Fluorescent Proteins / genetics HSP70 Heat-Shock Proteins / genetics Nerve Net / physiology* Neural Inhibition / genetics Organ Specificity / genetics Saccharomyces cerevisiae Proteins / biosynthesis Saccharomyces cerevisiae Proteins / genetics* Saccharomyces cerevisiae Proteins / metabolism Touch / genetics Touch / physiology Trans-Activators / genetics Transcription Factors / biosynthesis Transcription Factors / genetics* Transcription Factors / metabolism Zebrafish / embryology Zebrafish / genetics*
IF 9.504
Times Cited 240
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Zebrafish SAG?