Targeted transgene integration overcomes variability of position effects in zebrafish.

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.This work was funded by ‘BOLD’ Marie-Curie Initial Training Network; and ‘ZFHealth’ Integrating project in the Framework 7 programme of the European RESEARCH ARTICLE Development (2014) doi:10.1242/dev.100347Development Commission; University of Birmingham (F.M.); Temple University; and the National Institutes of Health (NIH) [HD061749 to D.B.]

Danio rerio (Hamilton, 1822) širdies ir kraujagyslių sistemos vystymosi ir regeneracinių mechanizmų tyrimai

Danio rerio (Hamilton, 1822) is a powerful vertebrate model system, which provides a unique combination of advantages that are important for an investigation of cardiovascular development and regeneration. However, conditional mutagenesis, which is essential for dissecting a role of developmental genes in regeneration, has not been demonstrated in the adult zebrafish. This remains a main disadvantage of D. rerio model system. The main aim of this research project was to use insertional gene trap alleles, tbx5atpl58 and fli1btpl50, to study cardiovascular development and regeneration in D. rerio. During this studies, the first conditional induction of the mutation was performed in the adult fish. Incubation of tbx5atpl58R;tnnt2a:CreERT2 adults in 4-hydroxytamoxifen induced a recombination between lox71 and lox66 sites, inverted the gene trap mutagenic cassette, and resulted in a successful re-mutation of tbx5atpl58R locus specifically in the heart. Generated conditional mutants with hearts mosaic for tbx5atpl58R/tpl58R, tbx5atpl58R/tpl58, and tbx5atpl58/tpl58 cardiomyocytes exhibited severe cardiac regeneration phenotypes. This indicated that tbx5a plays a critical role in zebrafish heart regeneration. Six new insertional genetrap mutant lines were characterized using iPCR and 5’RACE methods. One of the identified gene trap lines (fli1btpl50) that had a mutation in fli1b gene was found to undergo normal regeneration of the blood vessels, but exhibited GFP overexpression in vasculature and blood at 7 days after partial amputation of the caudal fin. This suggested that fli1b might be important for regeneration of vascular system, but it’s role is most likely being compensated by it’s paralog fli1a

Studying the mechanisms of cardiovascular development and regeneration in Danio rerio (Hamilton, 1822)

Danio rerio (Hamilton, 1822) is a powerful vertebrate model system, which provides a unique combination of advantages that are important for an investigation of cardiovascular development and regeneration. However, conditional mutagenesis, which is essential for dissecting a role of developmental genes in regeneration, has not been demonstrated in the adult zebrafish. This remains a main disadvantage of D. rerio model system. The main aim of this research project was to use insertional gene trap alleles, tbx5atpl58 and fli1btpl50, to study cardiovascular development and regeneration in D. rerio. During this studies, the first conditional induction of the mutation was performed in the adult fish. Incubation of tbx5atpl58R;tnnt2a:CreERT2 adults in 4-hydroxytamoxifen induced a recombination between lox71 and lox66 sites, inverted the gene trap mutagenic cassette, and resulted in a successful re-mutation of tbx5atpl58R locus specifically in the heart. Generated conditional mutants with hearts mosaic for tbx5atpl58R/tpl58R, tbx5atpl58R/tpl58, and tbx5atpl58/tpl58 cardiomyocytes exhibited severe cardiac regeneration phenotypes. This indicated that tbx5a plays a critical role in zebrafish heart regeneration. Six new insertional genetrap mutant lines were characterized using iPCR and 5’RACE methods. One of the identified gene trap lines (fli1btpl50) that had a mutation in fli1b gene was found to undergo normal regeneration of the blood vessels, but exhibited GFP overexpression in vasculature and blood at 7 days after partial amputation of the caudal fin. This suggested that fli1b might be important for regeneration of vascular system, but it’s role is most likely being compensated by it’s paralog fli1a

Internal epitope tagging informed by relative lack of sequence conservation

Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function

Analysis of a conditional gene trap reveals that <i>tbx5a</i> is required for heart regeneration in zebrafish

<div><p>The ability to conditionally inactivate genes is instrumental for fine genetic analysis of all biological processes, but is especially important for studies of biological events, such as regeneration, which occur late in ontogenesis or in adult life. We have constructed and tested a fully conditional gene trap vector, and used it to inactivate <i>tbx5a</i> in the cardiomyocytes of larval and adult zebrafish. We observe that loss of <i>tbx5a</i> function significantly impairs the ability of zebrafish hearts to regenerate after ventricular resection, indicating that Tbx5a plays an essential role in the transcriptional program of heart regeneration.</p></div