Abstract

We describe a conditional in vivo protein-trap mutagenesis system that reveals spatiotemporal protein expression dynamics and can be used to assess gene function in the vertebrate Danio rerio. Integration of pGBT-RP2.1 (RP2), a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with >97% knockdown of normal transcript amounts and simultaneously reports protein expression for each locus. The mutant alleles are revertible in somatic tissues via Cre recombinase or splice-site-blocking morpholinos and are thus to our knowledge the first systematic conditional mutant alleles outside the mouse model. We report a collection of 350 zebrafish lines that include diverse molecular loci. RP2 integrations reveal the complexity of genomic architecture and gene function in a living organism and can provide information on protein subcellular localization. The RP2 mutagenesis system is a step toward a unified 'codex' of protein expression and direct functional annotation of the vertebrate genome.

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Acknowledgements

The US National Institute on Drug Abuse (DA14546), US National Institute of General Medical Sciences (GM63904), National Institute of Diabetes and Digestive and Kidney Diseases (F30DK083219 and P30DK084567) and the Mayo Foundation provided funding for this research. S.S. and V.S. acknowledge funding support from the Council of Scientific and Industrial Research (grant FAC002), India. We thank members of the Center for Genome Engineering at the University of Minnesota for providing collaborative discussion and resources, the staff of the Zebrafish Core Facility at the Mayo Clinic for providing zebrafish care, D. Argue for programming the zfishbook website, E. Klee for bioinformatic analyses of the zebrafish transcriptome used in theoretical design and testing of the RP2 system, A. Person for assistance in injection of fluorescently labeled dextrans, and InSciEd Out participants and Summer Undergraduate Research Fellow, N. Boczek, for imaging RFP-expressing fish.

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Affiliations

  1. Mayo Clinic, Rochester, Minnesota, USA.

    • Karl J Clark
    • , Yonghe Ding
    • , Stephanie E Westcot
    • , Victoria M Bedell
    • , Tammy M Greenwood
    • , Mark D Urban
    • , Kimberly J Skuster
    • , Andrew M Petzold
    • , Jun Ni
    • , Xiaolei Xu
    •  & Stephen C Ekker
  2. University of Minnesota, Minneapolis, Minnesota, USA.

    • Darius Balciunas
    • , Stephanie E Westcot
    • , Andrew M Petzold
    • , Aubrey L Nielsen
    • , Sridhar Sivasubbu
    •  & Stephen C Ekker
  3. Temple University, Philadelphia, Pennsylvania, USA.

    • Darius Balciunas
  4. University of Cologne, Cologne, Germany.

    • Hans-Martin Pogoda
    •  & Matthias Hammerschmidt
  5. Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research, Delhi, India.

    • Ashok Patowary
    • , Vinod Scaria
    •  & Sridhar Sivasubbu

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Contributions

K.J.C., D.B., S.S. and S.C.E. designed the experiments. D.B. built R14, R15, R16, RP2 and pEF1a-cd99l2-RFP vectors. S.S. built pX. J.N. made the pEF1a-RFP vector. D.B. piloted the production of R14, R15 and R16 lines. K.J.C. trained and managed the team that produced >300 RP2 lines and molecularly characterized lines other than GBT0031, GBT0039 and GBT0043 (D.B.). M.D.U., T.M.G., A.L.N., K.J.S. and K.J.C. identified and propagated mRFP-expressing lines. A.M.P., M.D.U. and K.J.C. photographed mRFP expression patterns. K.J.S. and K.J.C. molecularly characterized lines by 5′ rapid amplification of cDNA ends, inverse PCR and quantitative PCR. T.M.G., K.J.C. and D.B. characterized the reversion of GBT0031 by Cre mRNA and morpholino injection. D.B. injected fluorescently conjugated dextran into GBT0046 and imaged its uptake. J.N. imaged GBT0043 fish and together with K.J.C. injected fish with EF1a-RFP and EF1a-cd99l2-RFP vectors. K.J.C. imaged the injected fish. S.E.W. analyzed the GBT0156/frasI phenotype and reversion. V.M.B. conducted in situ hybridizations. Y.D. identified GBT0348 in a screen performed in X.X.'s laboratory and contributed quantitative PCR data for three more RP2 lines. H.-M.P. tested the GBT protocol in M.H.'s lab and produced several independent lines, including the initial assessment and imaging of GBT0040. A.P., V.S. and S.S. produced and analyzed 3′ exon trap data for Supplementary Figure 2. K.J.C. and S.C.E. were primary authors of the text.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Stephen C Ekker.

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DOI

https://doi.org/10.1038/nmeth.1606

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