Targeting neural circuitry in zebrafish using GAL4 enhancer trapping

Scott, Ethan K.; Mason, Lindsay; Arrenberg, Aristides B.; Ziv, Limor; Gosse, Nathan J.; Xiao, Tong; Neil, C.; Asakawa, Kazuhide; Kawakami, Koichi; Baier, Herwig

doi:10.1038/nmeth1033

Cited by 371 publications

(393 citation statements)

References 12 publications

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“…However, the synthetic inducible promoter (GAL4) of the output gene used in this work may not always be a strong stable promoter in the tested cell lines. A high level of Gal4 expression can also cause toxicity to normal cells 27 . Moreover, this system is only a one-stage cascade.…”

Section: Discussionmentioning

confidence: 99%

Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells

et al. 2014

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The conventional strategy for cancer gene therapy offers limited control of specificity and efficacy. A possible way to overcome these limitations is to construct logic circuits. Here we present modular AND gate circuits based on CRISPR-Cas9 system. The circuits integrate cellular information from two promoters as inputs and activate the output gene only when both inputs are active in the tested cell lines. Using the luciferase reporter as the output gene, we show that the circuit specifically detects bladder cancer cells and significantly enhances luciferase expression in comparison to the human telomerase reverse transcriptase-renilla luciferase construct. We also test the modularity of the design by replacing the output with other cellular functional genes including hBAX, p21 and E-cadherin. The circuits effectively inhibit bladder cancer cell growth, induce apoptosis and decrease cell motility by regulating the corresponding gene. This approach provides a synthetic biology platform for targeting and controlling bladder cancer cells in vitro.

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Section: Discussionmentioning

confidence: 99%

Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells

et al. 2014

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“…Implementation of such methods, however, has been hampered by the lack of a transgenesis method that can generate random insertions efficiently throughout the genome. In addition, whereas the Gal4-VP16 fusion gene was used in zebrafish to overcome the weak gene expression activity of the full-length Gal4 (6), it has been known that expression of Gal4-VP16, a transcription factor with strong activator domains, causes nonspecific toxic effects in vertebrate cells (7,8) and in zebrafish (6,9). Hence, construction of a less toxic transcription activator would be advantageous for largescale screens in zebrafish.…”

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confidence: 99%

“…Recently, we reported a highly efficient transgenesis method and gene trap and enhancer trap methods by using the Tol2 transposon system (13,14). More recently, we and others have taken advantage of these methods to isolate fish lines expressing Gal4-VP16 in specific tissues (9,15). In the present study we aim to further develop methodologies in zebrafish that enable targeted expression of a desired gene in desired cells.…”

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confidence: 99%

Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish

Asakawa

Suster

Mizusawa

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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516

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Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P elementmediated 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.targeted gene expression ͉ Gal4-UAS ͉ tetanus toxin ͉ touch response ͉ interneuron

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“…Importantly, one of the most common methodologies ensuring cell-specific expression of transgenes in zebrafish is the Gal4-UAS binary system (derived from yeast), in which the transcription of genes placed 3 0 of an upstream activating sequence (UAS) relies on the DNA binding of the Gal4 transcriptional activator . Gene-and enhancer-trap methods have been applied to establish a significant number of Gal4 transgenic lines (Davison et al 2007;Scott and Baier 2009;Kawakami et al 2010;Balciuniene et al 2013), several of which are neural-specific (Scott et al 2007;. Notably, in these lines the Gal4 open reading frame (ORF) is randomly integrated in the fish genome through Tol2-based transposition, and the insertion site is not mapped; therefore, the sequence of the promoter elements driving Gal4 expression is unknown.…”

Section: Crispr/cas9 and Gal4/uas Combination For Cell-specific Gene mentioning

confidence: 99%

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish

Albadri

Santis

Donato

et al. 2017

Research and Perspectives in Neurosciences

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The zebrafish (Danio rerio) has emerged in recent years as a powerful vertebrate model to study neuronal circuit development and function, thanks to its relatively small size, rapid external development and translucency. These features allow the easy application of in vivo microscopy analysis and optical perturbation of neuronal function. So far, genetic manipulation in zebrafish has been limited to the generation of constitutive loss-of-function alleles and transgenic models.

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Targeting neural circuitry in zebrafish using GAL4 enhancer trapping

Cited by 371 publications

References 12 publications

Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells

Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells

Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish

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