Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron

Hardy, Melissa; Ross, Louis; Chien, Chi-­Bin

doi:10.1002/dvdy.21318

Cited by 32 publications

(19 citation statements)

References 20 publications

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“…However, laser induced heat-shocks have successfully been used in Drosophila (Halfon et al 1997), butterfly (Ramos et al 2006) and zebrafish (Halloran et al 2000) and thus should be applicable to Xenopus as well. Even the use of local activation by a modified soldering iron seems feasible (Hardy et al 2007). Alternatively, the establishment of effector strains with tissue-restricted promoters could be used.…”

Section: Discussionmentioning

confidence: 99%

Heat-shock inducible Cre strains to study organogenesis in transgenic Xenopus laevis

et al. 2009

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The frog Xenopus is a well established vertebrate model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a whole series of methods. We have expanded these approaches by establishing two transgenic Xenopus strains that allow specific interference with the activity of defined genes using a heat-shock inducible Cre recombinase that can induce upon heat-shock expression of a reporter gene in crossings to a corresponding reporter strain. We have applied this binary technique of gene interference in Xenopus development to overexpress the mutated HNF1 beta transcription factor at distinct developmental stages. Induction of HNF1 beta P328L329del by heat-shock at the gastrula stage resulted in a dramatic phenotype including malformation of the pronephros, gut, stomach, abnormal tail development and massive edemas indicative for kidney dysfunction. Thus, we have established the first binary inducible gene expression system in Xenopus laevis that can be used to study organogenesis.

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Section: Discussionmentioning

confidence: 99%

Heat-shock inducible Cre strains to study organogenesis in transgenic Xenopus laevis

et al. 2009

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“…Other approaches to locally misexpressing genes such as focal UV uncaging and localized heatshock tools (laser or soldering iron), do offer alternative ways to manipulate the expression of genes and markers in a small cluster of cells [7][8][9] ; however, cell transplantation establishes a final environment of analysis that is free of any trauma induced by UV, laser or heat making cell transplantation a more controlled experimental approach. In conclusion, we have found cell transplantation procedures to be a crucial part of our neurodevelopmental biology research.…”

Section: Discussionmentioning

confidence: 99%

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Deschene¹,

Barresi²

2009

JoVE

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Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered. Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions. Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes. Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras. We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another. We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures. In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo. These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology. Video LinkThe video component of this article can be found at https://www.jove.com/video/1422/ Protocol Part 1: Alexa 594 Labeled Embryos Microinjection Plate and Needle Preparation Making microinjection plates1. Prepare injection plates comprised of three to five 1-mm troughs within an agarose mold using a technique by Westerfield, 2007 1 . These troughs will snuggly hold the embryos in a line for efficient and systematic microinjections. Prior to making the plates, take three 1-mm, 4-inch long non-filament capillaries and carefully break them in half. Using superglue, glue two of these 2-inch long capillaries side-by-side on a flat surface. Glue the third capillary on top of these two, nestled in the grove, creating a pyramid shape with the three capillaries. Allow to dry and repeat until enough "trough molds" are constructed. 2. Pour 20-25mL (5mm depth) of molten 1.5% agarose in embryo medium (EM) [5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2 , 0.33mM MgSO 4 and 0.00003% methylene blue] into a 100mm petri dish. Immediately place three to five of the previously made pyramidshaped trough molds to the botto...

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“…Furthermore, a method using a modified soldering iron has been developed to enable a sitespecific heat induction in order to achieve spatial specificity of transgene control as well as temporal specificity [20,38].…”

Section: Heat Shock Promotersmentioning

confidence: 99%

Reverse genetics tools in zebrafish: A forward dive into endocrinology

Sugano

2013

General and Comparative Endocrinology

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The zebrafish is a powerful genetic model organism. In recent years, zebrafish has been increasingly used to model human diseases. Due to a number of recent technological advancements, the genetic tool box is now also stocked with sophisticated transgenic and reverse genetic tools. Here, we focus on both commonly used and recently established reverse genetic and transgenic tools available in zebrafish. These new developments make the zebrafish an even more attractive animal model in comparative endocrinology.

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Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron

Abstract: Misexpression of genes in a temporally

Cited by 32 publications

References 20 publications

Heat-shock inducible Cre strains to study organogenesis in transgenic Xenopus laevis

Heat-shock inducible Cre strains to study organogenesis in transgenic Xenopus laevis

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Reverse genetics tools in zebrafish: A forward dive into endocrinology

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