Tol2 Transgenesis Service in Zebrafish
ZeClinics' team of expert scientists can tailor our zebrafish Tol2 transgenesis services to address your unique research objectives.
We can introduce exogenous DNA randomly into the zebrafish genome to fastly generate custom genetic modification in transgenic zebrafish lines.
Why Choose ZeClinics's Zebrafish Tol2 Transgenesis Service?
Zebrafish Tol2 transgenesis is an exceptional technique that enables researchers to introduce precise genetic modifications into zebrafish, a alternative animal model in biomedical studies.
Using the Tol2 transgenesis system, we can insert large cargo or expression cassettes (like transcriptional reporters, coding sequences of human genes, fluorescent reporters, sensors to test environmental changes) under the control of a promoter of interest. This strategy is often used for tissue-specific reporter expression or to overexpress mutant proteins.
With our advanced Zebrafish Tol2 transgenesis service, we generate transgenic zebrafish lines featuring desired genetic alterations, enabling unequaled insights into human health and disease.
Applications
- Transcriptional reporters
- Models to control tissue-specific gene expression
- Models to control the timing of gene expression
- Models to control or modify cellular activities (optogenetic and chemogenetic tools)
- Create Gal4 or Cre-recombinase driver lines
- Humanized zebrafish models
Advantages
Highly efficient integration of transgenes
Fast genome edits
Possibility to finely control gene expression thanks to the presence of an extremely rich library of promoter sequences
Large-insert integration
Tol2 Transgenesis Method Description
Zebrafish transgenic lines are generated through a strategy based on the activity of the tol2 transposase. The Tol2 transposon system employs a specific DNA sequence known as the Tol2 transposon, capable of relocating or transposing itself within the zebrafish genome. This mechanism facilitates the stable and inheritable integration of transgenes into the genome.
One-cell-stage zebrafish embryos are injected with a mix containing tol2 mRNA and a plasmid containing the transgene of interest between two Tol2 sites. During the early stages of embryonic development, the Tol2 catalyzes the integration of the exogenous DNA into the genome of injected embryos. If this process happens in cells that give rise to the germline, injected fish will be able to transmit the transgene to their progeny, thus allowing the establishment of a transgenic line.
Figure 1. Generation of stable transgenic zebrafish line using the Tol2 transgenesis methodology. The Tol 2 technology allows random integration of the transgene of interest into the zebrafish genome. Upon injection of tol2 mRNA and a donor plasmid containing the desired sequence in one-cell stage embryos, mosaic integration of the sequence flanked by Tol2 recombination sites into the fish genome is achieved. When adulthood is reached, Founder fish carrying the transgene in the germline are identified and F1 is used for further phenotyping analysis.
Zebrafish Transgenesis Service Options
F0 Transgenic Mosaic
Select this option if you desire to delegate the design of a highly efficient transgenesis plasmid and acquire F0 transgenic larvae prepared to enter in your facility. This choice is ideal for researchers seeking to entrust skilled genome editing experts with the most intricate stages of the zebrafish transgenesis process.
Experimental steps
- Designed of the transgenesis plasmid by an experienced genetic engineer
- Synthesis of the designed vector
- In vivo evaluation of the transgenesis efficiency of the plasmid
- Injection of the validated plasmid into one-cell-stage embryos
- Batch of injected larvae to grow in your own facility
F1 Transgenic Larvae (Stable transgenic line)
Choose this option if you wish for us to provide the offspring of a validated founder (F1 larvae). This option is particularly advantageous for researchers who, in addition to benefiting from our expertise in designing an efficient transgenesis approach, aim to delegate the labor-intensive phase of founder screening and identification.
Experimental steps
- Steps listed in “F0 TRANSGENIC MOSAIC” plus:
- Growth of injected F0 larvae until adulthood
- Founder screening and identification of a fish carrying the desired transgene in its germline cells
- Breeding of the selected founder
- Maintenance of the selected founder for 4 months
Deliverables
- Batch of F1 larvae to grow and genotype in your own facility
F2 Transgenic Larvae (Stable transgenic line)
Choose this option if you want to rely on our vast expertise to conduct the entire process of establishing a new transgenic line.
Experimental steps
- Steps listed in “F1 TRANSGENIC LARVAE” plus:
- Growth of F1 larvae until adulthood
- Inbreeding of adult F1
- Maintenance of the F1 for 4 months
Deliverables
- Batch of F2 larvae to grow in your own facility
We'd like to hear from you
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services or have any other questions, please
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References
- Suster ML, Kikuta H, Urasaki A, Asakawa K, Kawakami K. Transgenesis in zebrafish with the tol2 transposon system. Methods Mol Biol. 2009;561:41-63.
- Kawakami K, Asakawa K, Muto A, Wada H. Tol2-mediated transgenesis, gene trapping, enhancer trapping, and Gal4-UAS system. Methods Cell Biol. 2016;135:19-37.
- Pakula A, Lek A, Widrick J, Mitsuhashi H, Bugda Gwilt KM, Gupta VA, Rahimov F, Criscione J, Zhang Y, Gibbs D, Murphy Q, Manglik A, Mead L, Kunkel L. Transgenic zebrafish model of DUX4 misexpression reveals a developmental role in FSHD pathogenesis. Hum Mol Genet. 2019 Jan 15;28(2):320-331.