ZeGenesis – Genetic Services

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transgenic-lines-icon Transgenic Lines

The generation of a zebrafish transgenic line allows the expression of exogenous coding sequences (CDS) (e.g. transcription factors, CDS of human genes, fluorescent reporters, sensors to test environmental changes, optogenetic tools) under the control of tissue-specific promoters.

Applications

  • Stable overexpression of human genes´ Coding Sequences
  • Establishment of customized reporter lines (e.g. cell-specific labelling and in vivo protein or organelles localization)
  • Generation of genetic tools to study biological processes (e.g. pathway modulation)
  • Creation of environmental sensors, optogenetic and chemogenetic tools

Advantages

Highly efficient integration of transgenes

Possibility to finely control gene expression thanks to the presence of an extremely rich library of promoter sequences 

Capability to generate a high number of economic and highly informative genetic lines 

Due to its optical transparency, zebrafish is ideal for models involving luminescence, such as reporter and optogenetic lines

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

  • Heterozygous transgenic embryos (F1)
  • Homozygous transgenic embryos (F2)

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.