NDLI: Targeted transgene integration overcomes variability of position effects in zebrafish.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Roberts, Jennifer Anne Miguel-Escalada, Irene Slovik, Katherine Joan Walsh, Kathleen Theodora Hadzhiev, Yavor Sanges, Remo Stupka, Elia Marsh, Elizabeth Kate Balciuniene, Jorune Balciunas, Darius Müller, Ferenc
Description	Country affiliation: United kingdom Author Affiliation: Roberts JA ( School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, B15 2TT, UK.)
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.
File Format	HTM / HTML
ISSN	09501991
e-ISSN	14779129
DOI	10.1242/dev.100347
Journal	Development
Issue Number	3
Volume Number	141
Language	English
Publisher	The Company of Biologists
Publisher Date	2014-02-01
Publisher Place	Great Britain (UK)
Access Restriction	Open
Subject Keyword	Discipline Developmental Discipline Biology Chromosomal Position Effects Genetics Gene Targeting Mutagenesis, Insertional Transgenes Zebrafish Animals Animals, Genetically Modified Brain Metabolism Enhancer Elements, Genetic Gene Expression Regulation Gene Transfer Techniques Genes, Reporter Genetic Loci Genome Integrases Lens, Crystalline Molecular Sequence Data Reproducibility Of Results Xenopus Laevis Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Developmental Biology Molecular Biology

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