Targeted transgene integration overcomes variability of position effects in zebrafish

Abstract

Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish. KEY WORDS: Zebrafish, Integrase, Transgenesis, Tol2, Enhancer, Position effects INTRODUCTIONTransgenesis is one of the fastest growing technologies in the zebrafish model system. ZFIN, the Zebrafish Model Organism Database (Bradford et al., 2011), lists 10,867 transgenic zebrafish lines. Transgenic zebrafish are produced for a variety of reasons, from in vivo labelling of cells and tissues to tissue-specific cell ablation, analysis of gene function, protein dynamics and localisation, generation of disease models through mis-expression of diseaseassociated genes, or manipulation of gene activities for developmental and physiological analysis (e.g. Gilmour et al., 2002;Langenau et al., 2005; Curado et al., 2008;Wyart et al., 2009;Hans et al., 2011). RESEARCH ARTICLE TECHNIQUES AND RESOURCESWith the publication of the ENCODE project and the discovery of the pervasiveness of predicted non-coding cis-regulatory modules (CRMs) in vertebrate genomes, emphasis is put on the functional validation of these computationally and biochemically predicted elements. Small lab...

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