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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.

SUBMITTER: Roberts JA

PROVIDER: S-EPMC3899822 | biostudies-other | 2014 Feb

REPOSITORIES: biostudies-other

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Targeted transgene integration overcomes variability of position effects in zebrafish.

Roberts Jennifer Anne JA Miguel-Escalada Irene I Slovik Katherine Joan KJ Walsh Kathleen Theodora KT Hadzhiev Yavor Y Sanges Remo R Stupka Elia E Marsh Elizabeth Kate EK Balciuniene Jorune J Balciunas Darius D Müller Ferenc F

Development (Cambridge, England) 20140201 3

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 ...[more]

PMID: 24449846

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Project description:The brain generally integrates a multitude of sensory signals to form a unified percept. Even in cursor control tasks, such as reaching while looking at rotated visual feedback on a monitor, visual information on cursor position and proprioceptive information on hand position are partially integrated (sensory coupling), resulting in mutual biases of the perceived positions of cursor and hand. Previous studies showed that the strength of sensory coupling (sum of the mutual biases) depends on the experience of kinematic correlations between hand movements and cursor motions, whereas the asymmetry of sensory coupling (difference between the biases) depends on the relative reliabilities (inverse of variability) of hand-position and cursor-position estimates (reliability rule). Furthermore, the precision of movement control and perception of hand position are known to differ between hands (left, right) and workspaces (ipsilateral, contralateral), and so does the experience of kinematic correlations from daily life activities. Thus, in the present study, we tested whether strength and asymmetry of sensory coupling for the endpoints of reaches in a cursor control task differ between the right and left hand and between ipsilateral and contralateral hemispace. No differences were found in the strength of sensory coupling between hands or between hemispaces. However, asymmetry of sensory coupling was less in ipsilateral than in contralateral hemispace: in ipsilateral hemispace, the bias of the perceived hand position was reduced, which was accompanied by a smaller variability of the estimates. The variability of position estimates of the dominant right hand was also less than for the non-dominant left hand, but this difference was not accompanied by a difference in the asymmetry of sensory coupling - a violation of the reliability rule, probably due a stronger influence of visual information on right-hand movements. According to these results, the long-term effects of the experienced kinematic correlation between hand movements and cursor motions on the strength of sensory coupling are generic and not specific for hemispaces or hands, whereas the effects of relative reliabilities on the asymmetry of sensory coupling are specific for hemispaces but not for hands.

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Targeted transgene integration overcomes variability of position effects in zebrafish.

Publications

Targeted transgene integration overcomes variability of position effects in zebrafish.

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