Abstract
Evaluating the biological relevance of the myriad putative regulatory noncoding sequences in vertebrate genomes represents a huge challenge. Functional analyses in vivo have typically relied on costly and labor-intensive transgenic strategies in mice. Transgenesis has also been applied in nonrodent vertebrates, such as zebrafish, but until recently these efforts have been hampered by significant mosaicism and poor rates of germline transmission. We have developed a transgenic strategy in zebrafish based on the Tol2 transposon, a mobile element that was recently identified in another teleost, Medaka. This method takes advantage of the increased efficiency of genome integration that is afforded by this intact DNA transposon, activity that is mediated by the corresponding transposase protein. The approach described in this protocol uses a universal vector system that permits rapid incorporation of DNA that is tagged with sequence targets for site-specific recombination. To evaluate the regulatory potential of a candidate sequence, the desired interval is PCR-amplified using sequence-specific primers that are flanked by the requisite target sites for cloning, and recombined into a universal expression plasmid (pGW_cfosEGFP). Purified recombinant DNAs are then injected into 1–2-cell zebrafish embryos and the resulting reporter expression patterns are analyzed at desired timepoints during development. This system is amenable to large-scale application, facilitating rapid functional analysis of noncoding sequences from both mammalian and teleost species.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kimura, M. & Ota, T. Protein polymorphism as a phase of molecular evolution. Nature 229, 467–469 (1971).
Pennacchio, L.A. & Rubin, E.M. Genomic strategies to identify mammalian regulatory sequences. Nature Rev. Genet. 2, 100–109 (2001).
Waterston, R.H. et al. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002).
Woolfe, A. et al. Highly conserved noncoding sequences are associated with vertebrate development. PLoS Biol 3, e7 (2005).
Koga, A., Suzuki, M., Inagaki, H., Bessho, Y. & Hori, H. Transposable element in fish. Nature 383, 30 (1996).
Davidson, A.E. et al. Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev. Biol. 263, 191–202 (2003).
Ivics, Z., Hackett, P.B., Plasterk, R.H. & Izsvak, Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510 (1997).
Thermes, V. et al. I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118, 91–98 (2002).
Kawakami, K. et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell 7, 133–144 (2004).
Dorsky, R.I., Sheldahl, L.C. & Moon, R.T. A transgenic Lef1/β-catenin-dependent reporter is expressed in spatially restricted domains throughout zebrafish development. Dev. Biol. 241, 229–237 (2002).
Fisher, S., Grice, E.A., Vinton, R.M., Bessling, S.L. & McCallion, A.S. Conservation of RET regulatory function from human to zebrafish without sequence similarity. Science 312, 276–279 (2006).
Johnson, S.L. & Zon, L.I. Genetic backgrounds and some standard stocks and strains used in zebrafish developmental biology and genetics. Methods Cell Biol 60, 357–359 (1999).
Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 (2005).
Frazer, K.A., Pachter, L., Poliakov, A., Rubin, E.M. & Dubchak, I. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 32, W273–W279 (2004).
Schwartz, S. et al. PipMaker — a web server for aligning two genomic DNA sequences. Genome Res. 10, 577–586 (2000).
Margulies, E.H., Blanchette, M., Haussler, D. & Green, E.D. Identification and characterization of multi-species conserved sequences. Genome Res. 13, 2507–2518 (2003).
Kolbe, D. et al. Regulatory potential scores from genome-wide three-way alignments of human, mouse, and rat. Genome Res. 14, 700–707 (2004).
Brudno, M., Chapman, M., Gottgens, B., Batzoglou, S. & Morgenstern, B. Fast and sensitive multiple alignment of large genomic sequences. BMC Bioinformatics 4, 66 (2003).
Westerfield, M. (ed.) The Zebrafish Book (University of Oregon Press, Eugene, OR, 1995).
Acknowledgements
This work was supported by a Basil O'Conner starter scholar award from the March of Dimes (A.S.M.) and grant GM071648 from the NIGMS (A.S.M.).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Fisher, S., Grice, E., Vinton, R. et al. Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nat Protoc 1, 1297–1305 (2006). https://doi.org/10.1038/nprot.2006.230
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.230
This article is cited by
-
Genome-wide analysis of cis-regulatory changes underlying metabolic adaptation of cavefish
Nature Genetics (2022)
-
Chromatin conformation of human oral epithelium can identify orofacial cleft missing functional variants
International Journal of Oral Science (2022)
-
Transgenic fluorescent zebrafish lines that have revolutionized biomedical research
Laboratory Animal Research (2021)
-
Distinct tooth regeneration systems deploy a conserved battery of genes
EvoDevo (2021)
-
Identification of downstream effectors of retinoic acid specifying the zebrafish pancreas by integrative genomics
Scientific Reports (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.