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.
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Method Article
The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach
In This Article
Summary
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 ....
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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 bmp4st72;smad5b1100 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.
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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.......
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Discussion
Zebrafish are ideally suited for studying the impact of ethanol exposure on development and disease states7,8. 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 contexts19,20. These strengths, combined with the strong development.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
The research presented in this article was supported by a grant from the National Institutes of Health/National Institute on Alcohol Abuse (NIH/NIAAA) R00AA023560 to C.B.L.
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Materials
| 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 |
References
- Denny, L., Coles, S., Blitz, R. Fetal alcohol syndrome and fetal alcohol spectrum disorders. American Family Physician. 96 (8), 515-522 (2017).
- Popova, S., et al. Comorbidity of fetal alcohol spectrum disorder: A systematic review and meta-analysis.
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