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Effective targeted gene ‘knockdown’ in zebrafish

Abstract

The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted ‘knockdown’ technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides1 (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode7, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene8 result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.

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Figure 1: Effective delivery and ubiquitous gene inhibition by injected morpholinos (MO).
Figure 2: Zygotic and maternal gene targeting by antisense morpholinos.
Figure 3: Post-somitogenesis embryonic targeting by antisense morpholinos in zebrafish.
Figure 4: The zebrafish as an effective model for the role of sonic hedgehog signalling in HPE.

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Acknowledgements

We thank S. Bingham, A. Chandrasekhar and M. Mieda for communication of results before publication; D. Mohn and P. Hackett for the E-line strain; A. Davidson, K. Finley and K. Markley for homozygous E-line fish; M. Halpern for the oep template DNA; A. Davidson, A. Kattan, K. Finley and C. Saunders for independently corroborating this method; P. Ingham for the ptc probe; S. Schulte-Merker for anti-Ntl antibody; J. Larson for technical support; M. Kofron, J. Heasman, J. Summerton and P. Morcos for sharing ideas on the possible uses of morpholinos as antisense inhibitors; and the zebrafish community for their suggestions and encouragement.

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Correspondence to Stephen C. Ekker.

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Nasevicius, A., Ekker, S. Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26, 216–220 (2000). https://doi.org/10.1038/79951

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