Dataset Information

Expressional artifact caused by a co-injection marker rol-6 in C. elegans.

ABSTRACT: In transgenic research, selection markers have greatly facilitated the generation of transgenic animals. A prerequisite for a suitable selection marker to be used along with a test gene of interest is that the marker should not affect the phenotype of interest in transformed animals. One of the most common selection markers used in C. elegans transgenic approaches is the rol-6 co-injection marker, which induces a behavioral roller phenotype due to a cuticle defect but is not known to have other side effects. However, we found that the rol-6 co-injection marker can cause expression of GFP in the test sequence in a male-specific interneuron called CP09. We found that the rol-6 gene sequence included in the marker plasmid is responsible for this unwanted expression. Accordingly, the use of the rol-6 co-injection marker is not recommended when researchers intend to examine precise expression or perform functional studies especially targeting male C. elegans neurons. The rol-6 sequence region we identified can be used to drive a specific expression in CP09 neuron for future research.

SUBMITTER: Jin H

PROVIDER: S-EPMC6892501 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Expressional artifact caused by a co-injection marker rol-6 in C. elegans.

Jin HoYong H Emmons Scott W SW Kim Byunghyuk B

PloS one 20191204 12

In transgenic research, selection markers have greatly facilitated the generation of transgenic animals. A prerequisite for a suitable selection marker to be used along with a test gene of interest is that the marker should not affect the phenotype of interest in transformed animals. One of the most common selection markers used in C. elegans transgenic approaches is the rol-6 co-injection marker, which induces a behavioral roller phenotype due to a cuticle defect but is not known to have other ...[more]

PMID: 31800569

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Project description:Optical mapping is a high-resolution fluorescence imaging technique, which provides highly detailed visualizations of the electrophysiological wave phenomena, which trigger the beating of the heart. Recent advancements in optical mapping have demonstrated that the technique can now be performed with moving and contracting hearts and that motion and motion artifacts, once a major limitation, can now be overcome by numerically tracking and stabilizing the heart's motion. As a result, the optical measurement of electrical activity can be obtained from the moving heart surface in a co-moving frame of reference and motion artifacts can be reduced substantially. The aim of this study is to assess and validate the performance of a 2D marker-free motion tracking algorithm, which tracks motion and non-rigid deformations in video images. Because the tracking algorithm does not require markers to be attached to the tissue, it is necessary to verify that it accurately tracks the displacements of the cardiac tissue surface, which not only contracts and deforms, but also fluoresces and exhibits spatio-temporal physiology-related intensity changes. We used computer simulations to generate synthetic optical mapping videos, which show the contracting and fluorescing ventricular heart surface. The synthetic data reproduces experimental data as closely as possible and shows electrical waves propagating across the deforming tissue surface, as seen during voltage-sensitive imaging. We then tested the motion tracking and motion-stabilization algorithm on the synthetic as well as on experimental data. The motion tracking and motion-stabilization algorithm decreases motion artifacts approximately by 80% and achieves sub-pixel precision when tracking motion of 1-10 pixels (in a video image with 100 by 100 pixels), effectively inhibiting motion such that little residual motion remains after tracking and motion-stabilization. To demonstrate the performance of the algorithm, we present optical maps with a substantial reduction in motion artifacts showing action potential waves propagating across the moving and strongly deforming ventricular heart surface. The tracking algorithm reliably tracks motion if the tissue surface is illuminated homogeneously and shows sufficient contrast or texture which can be tracked or if the contrast is artificially or numerically enhanced. In this study, we also show how a reduction in dissociation-related motion artifacts can be quantified and linked to tracking precision. Our results can be used to advance optical mapping techniques, enabling them to image contracting hearts, with the ultimate goal of studying the mutual coupling of electrical and mechanical phenomena in healthy and diseased hearts.

Dataset Information

Expressional artifact caused by a co-injection marker rol-6 in C. elegans.

Publications

Expressional artifact caused by a co-injection marker rol-6 in C. elegans.

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