The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Summary

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

Abstract

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

Introduction

Prenatal ethanol exposure gives rise to a continuum of structural deficits and cognitive impairments termed fetal alcohol spectrum disorders (FASD)^1,2,3,4. The complex relationships between multiple factors make studying and understanding the etiology of FASD in humans challenging. To resolve this challenge, a wide variety of animal models have been used. The biological and experimental tools available in these models have proven crucial in developing our understanding of the mechanistic basis of ethanol teratogenicity, and the results from ....

Protocol

All zebrafish embryos used in this procedure were raised and bred following established IACUC protocols¹². These protocols were approved by the University of Louisville.

NOTE: The wild-type zebrafish strain, AB, and the bmp4^st72;smad5^b1100 double mutant line were used in this study. All the water used in this procedure was sterile reverse osmosis water. Confocal images were taken under a laser-scanning confocal microscope. The endoderm measurements were made using the measure tool in ImageJ. All the statistical analyses were performed using statistical software.

Results

To generate the transgenic constructs, we used the Tol2 transgenesis system. Three entry vectors, including p5E, which holds the gene promoter/enhancer elements, pME, which holds the gene to be expressed by the promoter/enhancer elements, and p3E which, at minimum, holds the polyA tail, were used to generate the transgenic construct via multisite gateway LR cloning. The destination vector, pDest, provides the Tol2 repeats for the genomic insertion of the transgenic construct in zebrafish embryos and contains all.......

Discussion

Zebrafish are ideally suited for studying the impact of ethanol exposure on development and disease states^7,8. Zebrafish produce large numbers of translucent, externally fertilized, genetically tractable embryos, which allows for the live imaging of several transgene-labeled tissues and cell types simultaneously in multiple environmental contexts^19,20. These strengths, combined with the strong development.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Addgene Tol2 toolbox	https://www.addgene.org/kits/cole-tol2-neuro-toolbox/
Air			Provided directly by the university
Ampicillin	Fisher Scientific	BP1760
Analytical Balance	VWR	10204-962
Borosil 1.0 mm OD x 0.75 mm ID Capillary	FHC	30-30-0
Calcium Chloride	VWR	97062-590
Chloramphenicol	BioVision	2486
EDTA	Fisher Scientific	BP118-500
Fluorescent Dissecting Microscope	Olympus	SZX16
Kanamycin	Fisher Scientific	BP906
Laser Scanning Confocal Microscope	Olympus	Fluoview FV1000
Lawson Lab Donor Plasmid Prep	https://www.umassmed.edu/lawson-lab/reagents/lawson-lab-protocols/
LB Agar	Fisher Scientific	BP9724
LB Broth	Fisher Scientific	BP1426
Low-EEO/Multi-Purpose/Molecular Biology Grade Agarose	Fisher Scientific	BP160-500
LR Clonase II Plus Enzyme	Fisher Scientific	12538200
Magnesium Sulfate (Heptahydrate)	Fisher Scientific	M63-500
Micro Pipette holder	Applied Scientific Instrumentation	MIMPH-M-PIP
Microcentrifuge tube 0.5 mL	VWR	10025-724
Microcentrifuge tube 1.5 mL	VWR	10025-716
Micromanipulator	Applied Scientific Instrumentation	MM33
Micropipette tips 10 μL	Fisher Scientific	13611106
Micropipette tips 1000 μL	Fisher Scientific	13611127
Micropipette tips 200 μL	Fisher Scientific	13611112
mMESSAGE mMACHINE SP6 Transcription Kit	Fisher Scientific	AM1340
Mosimann Lab Tol2 Calculation Worksheet	https://www.protocols.io/view/multisite-gateway-calculations-excel-spreadsheet-8epv599p4g1b/v1
NanoDrop Spectrophotometer	NanoDrop	ND-1000
NcoI	NEB	R0189S
NotI	NEB	R0189S
Petri dishes 100 mm	Fisher Scientific	FB012924
Phenol Red sodium salt	Sigma Aldrich	P4758-5G
Pipetman L p1000L Micropipette	Gilson	FA10006M
Pipetman L p200L Micropipette	Gilson	FA10005M
Pipetman L p2L Micropipette	Gilson	FA10001M
Potassium Chloride	Fisher Scientific	P217-500
Potassium Phosphate (Dibasic)	VWR	BDH9266-500G
Pressure Injector	Applied Scientific Instrumentation	MPPI-3
QIAprep Spin Miniprep Kit	Qiagen	27106
Sodium Bicarbonate	VWR	BDH9280-500G
Sodium Chloride	Fisher Scientific	S271-500
Sodium Phosphate (Dibasic)	Fisher Scientific	S374-500
Stericup .22 µm vacuum filtration system	Millipore	SCGPU11RE
Tol2 Wiki Page	http://tol2kit.genetics.utah.edu/index.php/Main_Page
Top10 Chemically Competent E. coli	Fisher Scientific	C404010
Vertical Pipetter Puller	David Kopf Instruments	720
Zebrafish microinjection mold	Adaptive Science Tools	i34