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ZeGenesis – Genetic Services

transgenic-lines-icon Transgenic Lines

ZeClinics can introduce exogenous DNA randomly into the zebrafish genome to generate fast and tailored genome edits in transgenic lines.

Using the Tol2 transposon-based system, we can insert large cargo or expression cassettes (like transcriptional reporters, CDS 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.

Tol2 Transgenesis

Get the edit you want along with the support you need to start your transgenic project.

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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

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

Method description

Zebrafish transgenic lines are generated through a strategy based on the activity of the tol2 transposase. 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.

Deliverables

  • Plasmid sequence.
  • Pictures showing fluorescence in the location of interest.
  • F0 injected, F1 or F2 embryos
WT, wild-type; HET, heterozygous; HOM, homozygous.

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References

  1. 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.
  2. 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.
  3. 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.