Elsevier

Methods

Volume 49, Issue 3, November 2009, Pages 275-281
Methods

The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

https://doi.org/10.1016/j.ymeth.2009.01.004Get rights and content

Abstract

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.

Introduction

The yeast transcriptional activator Gal4 has a modular structure consisting of the DNA-binding domain and the transcriptional activation domain [1], [2]. Gal4 binds to its specific recognition sequence UAS (for upstream activating sequence) and activates transcription of target genes [3]. Since Gal4 expressed in particular tissues stimulates expression of a gene linked to UAS in a tissue-specific manner, the Gal4-UAS system has been employed to analyze gene functions in vivo in Drosophila. Namely, the P element-mediated enhancer trapping efficiently creates a number of fly lines expressing Gal4 in specific cells, and genes of interest are expressed in a spatially and temporally regulated fashion in the Gal4-expressing cells [4].

The zebrafish is a useful model for genetic studies of vertebrate systems. Several hundreds of fertilized eggs can be obtained from a single mating and a large number of adult fish can be maintained in a limited laboratory space. Due to these advantages, a variety of genetic approaches for investigating gene function have been carried out; i.e., chemical mutagenesis [5], [6], retroviral insertional mutagenesis [7], [8], [9], target-selected mutagenesis [10], [11], zinc-finger nuclease-based mutagenesis [12], [13] and morpholino knock-down [14]. In addition to these, targeted gene expression in specific tissues by using the Gal4-UAS system was described in zebrafish [15]. However, the usefulness of the Gal4-UAS method had been limited since construction of transgenic lines expressing Gal4 in various tissues and cells had been laborious and time-consuming, mainly because of the lack of an efficient transgenesis method.

We have been developing transposon techniques by using the Tol2 transposable element [16]. We cloned a cDNA encoding the transposase protein from the medaka fish Tol2 element and developed a two-component transposition system [17], [18]. When a plasmid carrying the Tol2 element is injected to zebrafish embryos with the Tol2 transposase mRNA synthesized in vitro, the Tol2 element is excised from the plasmid and integrated into the genome by the activity of the transposase [19]. Because of the high transposition efficiency in the germ line and the capacity to carry a large DNA fragment [19], [20], the Tol2-mediated transgenesis has become a popular method to create transgenic zebrafish and has been applied to gene trap and enhancer trap approaches [21], [22], [23].

Recently, the Tol2 transposon system was successfully applied to the Gal4 gene trap and enhancer trap methods in zebrafish [24], [25], [26], [27]. First, we constructed a novel Gal4 variant Gal4FF, which we think suitable for transcription activation in zebrafish. Second, we developed gene trap and enhancer trap constructs by using the Tol2 transposon vector and Gal4FF. Third, we constructed transgenic fish carrying fluorescent reporter genes downstream of UAS and performed screens to identify transgenic fish expressing Gal4 in specific tissues and cells. Finally, we constructed transgenic fish carrying an effector gene, in this case a gene for tetanus toxin, downstream of UAS, and demonstrated that our system can inhibit neural functions. Here we describe the protocols to perform the Gal4 gene trap and enhancer trap screens in zebrafish and to apply the Gal4 transgenic fish to the studies of vertebrate neural functions.

Section snippets

A novel transcriptional activator Gal4FF

We developed a novel transcriptional activator Gal4FF that contains the 147 amino acids DNA-binding domain from Gal4 and two tandem repeats of a 13 amino acids transcription activation module (PADALDDFDLDML) from VP16 (Fig. 1) [28], [29]. In previous studies [15], [24], [25], [30], [31], [32], the full-length Gal4 or Gal4-VP16 that contained the DNA binding domain and the VP16 activation domain was used for the Gal4-UAS system in zebrafish. We decided to employ Gal4FF since it is less toxic

Gal4FF gene trap and enhancer trap screens

A scheme for the Gal4FF gene trap and enhancer trap screening is shown in Fig. 3A. A plasmid DNA carrying T2KSAGFF or T2KhspGFF was injected into fertilized eggs with Tol2 transposase mRNA synthesized in vitro. In each microinjection experiment, 8–16 injected embryos were analyzed by the excision assay to confirm that transposition occurred in the injected embryos (Fig. 3A). When the excision product is detected in less than 70–80% of injected embryos, this indicates that transposase did not

The choice of Gal4FF

We employed Gal4FF to develop the Gal4-UAS transactivation system. Scheer and Campos-Ortega created transgenic fish by using the full-length Gal4 [15], and Koster and Fraser performed the transient gene expression analysis by using Gal4-VP16 since they thought that the full-length Gal4 was not strong enough in zebrafish [30]. Although we have not conducted an exact comparison between Gal4FF and other Gal4 variants from the view of the transactivation activity, we think the Gal4FF-UAS system is

Acknowledgements

This work was supported by a postdoctoral fellowship from the Japan Society for the Promotion of Science (to K.A.), a Sasakawa Scientific Research Grant from the Japan Science Society (to K.A.), NIH/NIGMS R01 GM069382, the National BioResource project and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

The Tol2 constructs and fish lines described here are available upon request.

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