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.
Bibliographical noteFunding Information:
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.