The Vertebrate Codex Gene Breaking Protein Trap Library For Genomic Discovery and Disease Modeling Applications

The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT alleles displayed strong loss of function phenotypes comparable to published mutants using other mutagenesis strategies (ryr1b, fras1, tnnt2a, edar and hmcn1). Using molecular assessment after positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex including novel expression annotation, identifying new functional roles of the vertebrate genome and generating a diverse collection of potential models of human disease

Tol2 Gene Trap Integrations in the Zebrafish Amyloid Precursor Protein Genes appa and aplp2 Reveal Accumulation of Secreted APP at the Embryonic Veins

Background—The single spanning transmembrane amyloid precursor protein (APP) and its proteolytic product, amyloid-beta (Aβ) peptide, have been intensely studied due to their role in the pathogenesis of Alzheimer’s disease. However, the biological role of the secreted ectodomain of APP, which is also generated by proteolytic cleavage, is less well understood. Here, we report Tol2 red fluorescent protein (RFP) transposon gene trap integrations in the zebrafish amyloid precursor protein a (appa) and amyloid precursor-like protein 2 (aplp2) genes. The transposon integrations are predicted to disrupt the appa and aplp2 genes to primarily produce secreted ectodomains of the corresponding proteins that are fused to RFP. Results—Our results indicate the Appa-RFP and Aplp2 fusion proteins are likely secreted from the central nervous system and accumulate in the embryonic veins independent of blood flow. Conclusions—The zebrafish appa and aplp2 transposon insertion alleles will be useful for investigating the biological role of the secreted form of APP

Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis

Endothelial tubulogenesis is a crucial step in the formation of functional blood vessels during angiogenesis and vasculogenesis. Here, we use in vivo imaging of living zebrafish embryos expressing fluorescent fusion proteins of β-Actin, α-Catenin, and the ERM family member Moesin1 (Moesin a), to define a novel cord hollowing process that occurs during the initial stages of tubulogenesis in intersegmental vessels (ISVs) in the embryo. We show that the primary lumen elongates along cell junctions between at least two endothelial cells during embryonic angiogenesis. Moesin1-EGFP is enriched around structures that resemble intracellular vacuoles, which fuse with the luminal membrane during expansion of the primary lumen. Analysis of silent heart mutant embryos shows that initial lumen formation in the ISVs is not dependent on blood flow; however, stabilization of a newly formed lumen is dependent upon blood flow. Zebrafish moesin1 knockdown and cell transplantation experiments demonstrate that Moesin1 is required in the endothelial cells of the ISVs for in vivo lumen formation. Our analyses suggest that Moesin1 contributes to the maintenance of apical/basal cell polarity of the ISVs as defined by adherens junctions. Knockdown of the adherens junction protein Ve-cadherin disrupts formation of the apical membrane and lumen in a cell-autonomous manner. We suggest that Ve-cadherin and Moesin1 function to establish and maintain apical/basal polarity during multicellular lumen formation in the ISVs

The Gene Sculpt Suite: a set of tools for genome editing

The discovery and development of DNA-editing nucleases (Zinc Finger Nucleases, TALENs, CRISPR/Cas systems) has given scientists the ability to precisely engineer or edit genomes as never before. Several different platforms, protocols and vectors for precision genome editing are now available, leading to the development of supporting web-based software. Here we present the Gene Sculpt Suite (GSS), which comprises three tools: (i) GTagHD, which automatically designs and generates oligonucleotides for use with the GeneWeld knock-in protocol; (ii) MEDJED, a machine learning method, which predicts the extent to which a double-stranded DNA break site will utilize the microhomology-mediated repair pathway; and (iii) MENTHU, a tool for identifying genomic locations likely to give rise to a single predominant microhomology-mediated end joining allele (PreMA) repair outcome. All tools in the GSS are freely available for download under the GPL v3.0 license and can be run locally on Windows, Mac and Linux systems capable of running R and/or Docker. The GSS is also freely available online at www.genesculpt.org

Rapid Tumor Induction in Zebrafish by TALEN-Mediated Somatic Inactivation of the Retinoblastoma1 Tumor Suppressor rb1

Investigating the in vivo role of tumor suppressor genes in cancer is technically challenging due to their essential requirement during early animal development. To address this bottleneck, we generated genetic mosaic adult zebrafish using TALEN genome editing and demonstrate somatic inactivation of the tumor suppressor retinoblastoma1 (rb1) induces tumorigenesis at high frequency. 11–33% of 1-cell stage embryos injected with TALEN mRNAs targeting rb1 exon 2 or 3 develop tumors beginning as early as 3.5 months of age. Lesions predominantly arise in the brain and show features of neuroectodermal-like and glial-like tumors. Mutant allele analysis is consistent with tumor initiation due to somatic inactivation of rb1, revealing a conserved role for rb1 in tumor suppression across vertebrates. In contrast to genetic mosaics, heterozygous rb1−/+ adults show no evidence of neoplasia, while homozygous mutant rb1−/− are larval lethal. This is the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish, and highlights the utility of site-specific nucleases to create genetic mosaic zebrafish for tumor suppressor gene discovery. Somatic inactivation with site-directed nucleases in zebrafish presents a rapid and scalable strategy to study tumor suppressor gene function in cancer

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems