Elsevier

Methods

Volume 39, Issue 3, July 2006, Pages 199-206
Methods

Transposon-mediated gene trapping in zebrafish

https://doi.org/10.1016/j.ymeth.2005.12.006Get rights and content

Abstract

The Tol2 transposon system can create chromosomal insertions in the zebrafish germ lineage very efficiently. We constructed a Tol2-based gene trap vector, T2KSAG, which contains a splice accepter, the GFP gene and the polyA signal. In the pilot screen for gene trapping using T2KSAG, we identified 38 fish lines expressing GFP in specific organs and tissues. In the SAGp53A line, GFP is expressed in the forebrain and midbrain, and the insertion of the gene trap construct captured a transcript of the kab gene encoding a zebrafish homolog of the human KARP (Ku86 autoantigen related protein)-binding protein (KAB). In the SAGm18B line, GFP is expressed in the central nervous system, and the insertion captured a transcript of a gene for succinyl CoA:3-oxoacid CoA-transferase (SCOT). Here, we describe how we performed the gene trap screen and characterized the gene trap insertions and will discuss the outcome of the pilot screen.

Introduction

Zebrafish has been used as a model animal to study vertebrate development by both forward and reverse genetics approaches. For forward genetics, mainly two approaches have been carried out. First, a number of mutations causing phenotypic defects in embryonic development have been isolated by chemical mutagenesis [1], [2], and the mutated genes have been identified by positional cloning [3]. Second, an insertional mutagenesis method using a pseudotyped retrovirus was developed [4], and more than three hundreds genes essential for embryogenesis have been identified [5]. Although these methodologies have disclosed a number of important developmental genes and pathways, maintenance of fish during a large-scale chemical mutagenesis, positional cloning and handling of a pseudotyped retrovirus are still laborious and cannot be performed in every laboratory. For embryological studies, a transgenesis method was established [6]. By taking advantage of the transparency of embryos, this method has been applied to create transgenic fish expressing GFP in specific organs [7], [8], which are extremely useful to visualize developmental processes. The transgenic frequency using conventional methods is however fairly low and typically one hundred fish (or sometimes even more) has to be injected, raised and mated to obtain a single transgenic fish line. In order to circumvent these problems, we have developed a transposon technology in zebrafish [9], [10], for review see [11].

The Tol2 element was isolated from the genome of the medaka fish Oryzias latipes [12]. We identified an autonomous member of this element, which carried a gene for an active transposase [13], and cloned the cDNA of the transposase gene [14]. To date, the Tol2 element is the only natural transposon in vertebrate for which an autonomous member has been identified. We coinjected plasmid DNA carrying a Tol2-vector and mRNA synthesized in vitro by using the cDNA as a template into fertilized eggs, and demonstrated that the transposase synthesized from the mRNA can catalyze transposition of the Tol2-vector from the plasmid to the genome in the zebrafish germ lineage [15]. The transgenic frequency was very high since about half of fish injected with the transposon-donor plasmid DNA and the transposase mRNA at the one cell stage transmitted the transposon insertions to the next generation [9]. Thus, the Tol2 transposon system should facilitate transgenesis in zebrafish.

We further aimed to develop a gene trap method using the Tol2 transposon system. We constructed a Tol2-based vector, T2KSAG, which contains a splice accepter, the GFP gene and the polyA signal, and performed a pilot screen for gene trapping [9]. In this manuscript, we will describe the details of our gene trap method. First, we will describe how we created and characterized the gene trap insertions by showing two examples, and then describe what we learned from the pilot screen.

Section snippets

Structure of the gene trap vector

A gene trap vector, T2KSAG, contains a splice acceptor from the rabbit β-globin gene, the GFP gene and the SV40 polyA signal (Fig. 1A). In Fig. 1 and the text below, the left-hand side of T2KSAG is called 3′ and the right-hand side is called 5′ according to the direction of the transposase gene [14]. The GFP reporter gene on T2KSAG should be expressed in the following two cases: (1) when the construct is inserted downstream of a promoter of an endogenous gene (Fig. 1B, top) and upstream of the

Characterization of the SAGp53A insertion

Two out of 162 F1 embryos from a founder injected with the T2KSAG plasmid and transposase mRNA expressed GFP in the forebrain and midbrain, and the putative insertion causing this expression pattern was named SAGp53A (Fig. 3A). The GFP-positive F1 embryos were raised, and analyzed by Southern blot hybridization. The F1 fish carried a single transposon insertion, indicating that it caused the observed GFP expression (Fig. 3B). Tail fins were obtained from these fish, and genomic DNA surrounding

Concluding remarks

  • (1)

    The Tol2 transposon system is useful for constructing transgenic fish. Since more than half of fish injected with the Tol2 transposon system transmit the insertions to the next generation, germline transmitting founder fish will typically be identified by screening 10 injected fish or less as compared to 10 times that number using the traditional approach of DNA injection.

  • (2)

    The Tol2 transposon system is applicable to gene trapping in zebrafish. Since 38 unique GFP expression patterns were

Acknowledgments

This work was supported by the National Institute of Health (GM69382) and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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