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1.  Silent Tyrosinemia Type I Without Elevated Tyrosine or Succinylacetone Associated with Liver Cirrhosis and Hepatocellular Carcinoma 
Human Mutation  2016;37(10):1097-1105.
ABSTRACT
Tyrosinemia type I (TYRSN1, TYR I) is caused by fumarylacetoacetate hydrolase (FAH) deficiency and affects approximately one in 100,000 individuals worldwide. Pathogenic variants in FAH cause TYRSN1, which induces cirrhosis and can progress to hepatocellular carcinoma (HCC). TYRSN1 is characterized by the production of a pathognomonic metabolite, succinylacetone (SUAC) and is included in the Recommended Uniform Screening Panel for newborns. Treatment intervention is effective if initiated within the first month of life. Here, we describe a family with three affected children who developed HCC secondary to idiopathic hepatosplenomegaly and cirrhosis during infancy. Whole exome sequencing revealed a novel homozygous missense variant in FAH (Chr15(GRCh38):g.80162305A>G; NM_000137.2:c.424A > G; NP_000128.1:p.R142G). This novel variant involves the catalytic pocket of the enzyme, but does not result in increased SUAC or tyrosine, making the diagnosis of TYRSN1 problematic. Testing this novel variant using a rapid, in vivo somatic mouse model showed that this variant could not rescue FAH deficiency. In this case of atypical TYRSN1, we show how reliance on SUAC as a primary diagnostic test can be misleading in some patients with this disease. Augmentation of current screening for TYRSN1 with targeted sequencing of FAH is warranted in cases suggestive of the disorder.
doi:10.1002/humu.23047
PMCID: PMC5108417  PMID: 27397503
fumarylacetoacetate hydrolase; FAH; tyrosinemia type I; TYRSN1; TYR I; newborn screening; whole exome sequencing; pediatric liver cancer
2.  Crosslinked, Glassy Styrenic Surfactants Stabilize Quantum Dots Against Environmental Extremes 
Journal of materials chemistry  2009;19(35):6324-6327.
Semiconductor, quantum dot (QD) nanoparticles (including CdSe/ZnS, CdTe/ZnS, and CdSe) were encapsulated within cross-linked shells of amphiphilic polystyrene-block-poly(acrylic acid) block copolymer. Transmission electron microscopy revealed that each particle was surrounded by a uniform, layer of copolymer, and that the average diameter of the resulting QD-core micelles was between 25 and 50 nm, depending on the conditions of particle assembly. Overall, we found that aqueous suspensions of these QDs were substantially more stable to heat and pH than particles with other surface preparations; we argue that the enhanced stability is due to the uniform, hydrophobic coating of polystyrene around each particle and the reinforcement of this layer by shell-cross-linking. The biocompatibility of these particles was investigated by microinjection of particle suspension into live zebrafish embryos. The particles permanently stained the fish vasculature, but did not interfere with the normal development of the fish. We propose that QDs encapsulated in cross-linked block-copolymer shells allow QDs to be used in biological or biotechnological protocols requiring harsh reaction conditions.
Graphical Abstract
Encapsulating semiconductor quantum dot nanoparticles within a shell of crosslinked, poly(styrene-block-acrylic acid) surfactant enhances the stability of the particles against the harsh environments of some methods in biology and biotechnology.
doi:10.1039/B902275A
PMCID: PMC5129847  PMID: 27917026
3.  A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene 
JCI insight  2016;1(14):e88797.
Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b(L)) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases.
doi:10.1172/jci.insight.88797
PMCID: PMC5023154  PMID: 27642634
4.  A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene 
JCI Insight  null;1(14):e88797.
Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b(L)) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases.
By conducting a mutagenesis-based modifier screening in adult zebrafish, DNAJB6 is identified as a new susceptibility gene in addition to 3 known cardiomyopathy genes.
doi:10.1172/jci.insight.88797
PMCID: PMC5023154  PMID: 27642634
5.  Active recombinant Tol2 transposase for gene transfer and gene discovery applications 
Mobile DNA  2016;7:6.
Background
The revolutionary concept of “jumping genes” was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize. Transposable elements (TEs) represent the most abundant component of many eukaryotic genomes. Mobile elements are a driving force of eukaryotic genome evolution. McClintock’s Ac, the autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam3 from snapdragon define an ancient and diverse DNA transposon superfamily named hAT. Other members of the hAT superfamily include the insect element Hermes and Tol2 from medaka. In recent years, genetic tools derived from the ‘cut’ and ‘paste’ Tol2 DNA transposon have been widely used for genomic manipulation in zebrafish, mammals and in cells in vitro.
Results
We report the purification of a functional recombinant Tol2 protein from E.coli. We demonstrate here that following microinjection using a zebrafish embryo test system, purified Tol2 transposase protein readily catalyzes gene transfer in both somatic and germline tissues in vivo. We show that purified Tol2 transposase can promote both in vitro cutting and pasting in a defined system lacking other cellular factors. Notably, our analysis of Tol2 transposition in vitro reveals that the target site preference observed for Tol2 in complex host genomes is maintained using a simpler target plasmid test system, indicating that the primary sequence might encode intrinsic cues for transposon integration.
Conclusions
This active Tol2 protein is an important new tool for diverse applications including gene discovery and molecular medicine, as well as for the biochemical analysis of transposition and regulation of hAT transposon/genome interactions. The measurable but comparatively modest insertion site selection bias noted for Tol2 is largely determined by the primary sequence encoded in the target sequence as assessed through studying Tol2 protein-mediated transposition in a cell-free system.
Electronic supplementary material
The online version of this article (doi:10.1186/s13100-016-0062-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13100-016-0062-z
PMCID: PMC4818426  PMID: 27042235
Tol2 transposase; hAT superfamily; Recombinant transposase protein; Zebrafish; Transposition site preference
6.  FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science 
Human Gene Therapy  2016;27(6):451-463.
Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.
doi:10.1089/hum.2015.172
PMCID: PMC4931509  PMID: 26854857
7.  Influenza Knowledge, Attitude, and Behavior Survey for Grade School Students: Design and Novel Assessment Methodology 
Journal of community health  2014;39(6):1231-1240.
Background
Despite the fact infectious diseases can spread readily in grade schools, few studies have explored prevention in this setting. Additionally, we lack valid tools for students to self-report knowledge, attitudes, and behaviors. As part of an ongoing study of a curriculum intervention to promote healthy behaviors, we developed and evaluated age-appropriate surveys to determine students’ understanding of influenza prevention.
Methods
Surveys were adapted from adolescent and adult influenza surveys and administered to students in grades 2–5 (ages 7–11) at two Rochester public schools. We assessed student understanding by analyzing percent repeatability of 20 survey questions and compared percent “Don’t Know” (DK) responses across grades, gender, and race. Questions thought to be ambiguous after early survey administration were investigated in student focus groups, modified as appropriate, and reassessed.
Results
The response rate across all surveys was > 87%. Survey questions were well understood; 17 of 20 questions demonstrated strong pre/post repeatability (> 70%). Only 1 question showed an increase in DK response for higher grades (p <.0001). Statistical analysis and qualitative feedback led to modification of 3 survey questions and improved measures of understanding in the final survey administration.
Conclusions
Grade-school students’ knowledge, attitudes and behavior toward influenza prevention can be assessed using surveys. Quantitative and qualitative analysis may be used to assess participant understanding and refine survey development for pediatric survey instruments. These methods may be used to assess the repeatability and validity of surveys to assess the impact of health education interventions in young children.
doi:10.1007/s10900-014-9884-0
PMCID: PMC4769434  PMID: 24859735
Community health education; child health behavior; survey design; influenza
8.  Using engineered endonucleases to create knockout and knockin zebrafish models 
Summary
Over the last few years, the technology to create targeted knockout and knockin zebrafish animals has exploded. We have gained the ability to create targeted knockouts through the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated system (CRISPR/Cas). Furthermore, using the high-efficiency TALEN system, we were able to create knockin zebrafish using a single-stranded DNA (ssDNA) protocol described here. Through the use of these technologies, the zebrafish has become a valuable vertebrate model and an excellent bridge between the invertebrate and mammalian model systems for the study of human disease.
doi:10.1007/978-1-4939-1862-1_17
PMCID: PMC4385738  PMID: 25408414
TALEN; Genome Engineering; Zebrafish; HDR
9.  The New and TALENted Genome Engineering Toolbox 
Circulation research  2013;113(5):571-587.
Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the post-genomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.
doi:10.1161/CIRCRESAHA.113.301765
PMCID: PMC3965580  PMID: 23948583
TALENs; zebrafish; iPS cells; genome engineering; cardiovascular disease modeling
10.  Lessons from morpholino-based screening in zebrafish 
Briefings in Functional Genomics  2011;10(4):181-188.
Morpholino oligonucleotides (MOs) are an effective, gene-specific antisense knockdown technology used in many model systems. Here we describe the application of MOs in zebrafish (Danio rerio) for in vivo functional characterization of gene activity. We summarize our screening experience beginning with gene target selection. We then discuss screening parameter considerations and data and database management. Finally, we emphasize the importance of off-target effect management and thorough downstream phenotypic validation. We discuss current morpholino limitations, including reduced stability when stored in aqueous solution. Advances in MO technology now provide a measure of spatiotemporal control over MO activity, presenting the opportunity for incorporating more finely tuned analyses into MO-based screening. Therefore, with careful management, MOs remain a valuable tool for discovery screening as well as individual gene knockdown analysis.
doi:10.1093/bfgp/elr021
PMCID: PMC3144740  PMID: 21746693
morpholinos; zebrafish; knockdown
11.  WNT5A Mutations in Patients with Autosomal Dominant Robinow Syndrome 
Robinow syndrome is a skeletal dysplasia with both autosomal dominant and autosomal recessive inheritance patterns. It is characterized by short stature, limb shortening, genital hypoplasia and craniofacial abnormalities. The etiology of dominant Robinow syndrome is unknown, however the phenotypically more severe autosomal recessive form of Robinow syndrome has been associated with mutations in the orphan tyrosine kinase receptor, ROR2, which has recently been identified as a putative WNT5A receptor. Here we show that two different missense mutations in WNT5A, which result in amino acid substitutions of highly conserved cysteines, are associated with autosomal dominant Robinow syndrome. One mutation has been found in all living affected members of the original family described by Meinhard Robinow and another in a second unrelated patient. These missense mutations result in decreased WNT5A activity in functional assays of zebrafish and Xenopus development. This work suggests that a WNT5A/ROR2 signal transduction pathway is important in human craniofacial and skeletal development, and that proper formation and growth of these structures is sensitive to variations in WNT5A function.
doi:10.1002/dvdy.22156
PMCID: PMC4059519  PMID: 19918918
12.  A Sequence-Based Variation Map of Zebrafish 
Zebrafish  2013;10(1):15-20.
Abstract
Zebrafish (Danio rerio) is a popular vertebrate model organism largely deployed using outbred laboratory animals. The nonisogenic nature of the zebrafish as a model system offers the opportunity to understand natural variations and their effect in modulating phenotype. In an effort to better characterize the range of natural variation in this model system and to complement the zebrafish reference genome project, the whole genome sequence of a wild zebrafish at 39-fold genome coverage was determined. Comparative analysis with the zebrafish reference genome revealed approximately 5.2 million single nucleotide variations and over 1.6 million insertion–deletion variations. This dataset thus represents a new catalog of genetic variations in the zebrafish genome. Further analysis revealed selective enrichment for variations in genes involved in immune function and response to the environment, suggesting genome-level adaptations to environmental niches. We also show that human disease gene orthologs in the sequenced wild zebrafish genome show a lower ratio of nonsynonymous to synonymous single nucleotide variations.
doi:10.1089/zeb.2012.0848
PMCID: PMC3629779  PMID: 23590399
13.  The CRISPR System—Keeping Zebrafish Gene Targeting Fresh 
Zebrafish  2013;10(1):116-118.
Abstract
We are entering a new era in our ability to modify and edit the genomes of model organisms. Zinc finger nucleases (ZFNs) opened the door to the first custom nuclease-targeted genome engineering in the late 1990s. However, ZFNs remained out of reach for most research labs because of the difficulty of production, high costs, and modest efficacy in many applications. Transcription activator-like effector nucleases (TALENs) were built upon a DNA binding system discovered in a group of plant bacterial pathogens and broadened custom nuclease technology, showing significant improvements in both targeting flexibility and efficiency. Perhaps most importantly, TALENs are open source and easy to produce, providing zebrafish laboratories around the world with affordable tools that can be made in-house rapidly, at low cost, and with reliably high activity. Now a new system for targeted genome engineering derived from the CRISPR/Cas system in eubacteria and archaea promises to simplify this process further. Together, these tools will help overcome many of the bottlenecks that have constrained gene targeting in zebrafish, paving the way for advanced genome engineering applications in this model teleost.
doi:10.1089/zeb.2013.9999
PMCID: PMC3629780  PMID: 23536990
14.  Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development, Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis 
PLoS ONE  2015;10(6):e0130688.
Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes—fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a—had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a – ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.
doi:10.1371/journal.pone.0130688
PMCID: PMC4482254  PMID: 26110643
15.  Detection of 1α,25-Dihydroxyvitamin D-Regulated miRNAs in Zebrafish by Whole Transcriptome Sequencing 
Zebrafish  2014;11(3):207-218.
Abstract
The sterol hormone, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), regulates gene expression and messenger RNA (mRNA) concentrations in zebrafish in vivo. Since mRNA concentrations and translation are influenced by micro-RNAs (miRNAs), we examined the influence of 1α,25(OH)2D3 on miRNA expression in zebrafish in vivo with whole transcriptome RNA sequencing, searched for miRNA binding sites in 1α,25(OH)2D3-sensitive genes, and performed correlation analyses between 1α,25(OH)2D3-sensitive miRNAs and mRNAs. In vehicle- and 1α,25(OH)2D3-treated, 7-day postfertilization larvae, between 282 and 295 known precursor miRNAs were expressed, and in vehicle- and 1α,25(OH)2D3-treated fish, between 83 and 122 novel miRNAs were detected. Following 1α,25(OH)2D3 treatment, 31 precursor miRNAs were differentially expressed (p<0.05). The differentially expressed miRNAs are predicted to potentially alter mRNAs for metabolic enzymes, transcription factors, growth factors, and Jak-STAT signaling. We verified the role of a 1α,25(OH)2D3-sensitive miRNA, miR125b, by demonstrating alterations in the concentrations of the mRNA of a 1α,25(OH)2D3-regulated gene, Cyp24a1, following transfection of renal cells with a miR125b miRNA mimic. Changes in the Cyp24a1 mRNA concentration by the miR125b miRNA mimic were associated with changes in the protein for Cyp24a1. Our data show that 1α,25(OH)2D3 regulates miRNA in zebrafish larvae in vivo and could thereby influence vitamin D-sensitive mRNA concentrations.
doi:10.1089/zeb.2013.0899
PMCID: PMC4050706  PMID: 24650217
16.  Etv2 and Fli1b Function Together as Key Regulators of Vasculogenesis and Angiogenesis 
Objective
The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis.
Approach and Results
Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites.
Conclusions
Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.
doi:10.1161/ATVBAHA.114.304768
PMCID: PMC4427907  PMID: 25722433
angiogenesis; ETS transcription factor; vasculogenesis; zebrafish
17.  Transposon tools hopping in vertebrates 
In the past decade, tools derived from DNA transposons have made major contributions to vertebrate genetic studies from gene delivery to gene discovery. Multiple, highly complementary systems have been developed, and many more are in the pipeline. Judging which DNA transposon element will work the best in diverse uses from zebrafish genetic manipulation to human gene therapy is currently a complex task. We have summarized the major transposon vector systems active in vertebrates, comparing and contrasting known critical biochemical and in vivo properties, for future tool design and new genetic applications.
doi:10.1093/bfgp/eln049
PMCID: PMC2722259  PMID: 19109308
transposon; gene delivery; gene discovery; gene transfer vectors; vertebrates
18.  TALEN Knockout of the PSIP1 Gene in Human Cells: Analyses of HIV-1 Replication and Allosteric Integrase Inhibitor Mechanism 
Journal of Virology  2014;88(17):9704-9717.
ABSTRACT
HIV-1 utilizes the cellular protein LEDGF/p75 as a chromosome docking and integration cofactor. The LEDGF/p75 gene, PSIP1, is a potential therapeutic target because, like CCR5, depletion of LEDGF/p75 is tolerated well by human CD4+ T cells, and knockout mice have normal immune systems. RNA interference (RNAi) has been useful for studying LEDGF/p75, but the potent cofactor activity of small protein residua can be confounding. Here, in human cells with utility for HIV research (293T and Jurkat), we used transcription activator-like effector nucleases (TALENs) to completely eradicate all LEDGF/p75 expression. We performed two kinds of PSIP1 knockouts: whole-gene deletion and deletion of the integrase binding domain (IBD)-encoding exons. HIV-1 integration was inhibited, and spreading viral replication was severely impaired in PSIP1−/− Jurkat cells infected at high multiplicity. Furthermore, frameshifting the gene in the first coding exon with a single TALEN pair yielded trace LEDGF/p75 levels that were virologically active, affirming the cofactor's potency and the value of definitive gene or IBD exon segment deletion. Some recent studies have suggested that LEDGF/p75 may participate in HIV-1 assembly. However, we determined that assembly of infectious viral particles is normal in PSIP1−/− cells. The potency of an allosteric integrase inhibitor, ALLINI-2, for rendering produced virions noninfectious was also unaffected by total eradication of cellular LEDGF/p75. We conclude that HIV-1 particle assembly and the main ALLINI mechanism are LEDGF/p75 independent. The block to HIV-1 propagation in PSIP1−/− human CD4+ T cells raises the possibility of gene targeting PSIP1 combinatorially with CCR5 for HIV-1 cure.
IMPORTANCE LEDGF/p75 dependence is universally conserved in the retroviral genus Lentivirus. Once inside the nucleus, lentiviral preintegration complexes are thought to attach to the chromosome when integrase binds to LEDGF/p75. This tethering process is largely responsible for the 2-fold preference for integration into active genes, but the cofactor's full role in the lentiviral life cycle is not yet clear. Effective knockdowns are difficult because even trace residua of this tightly chromatin-bound protein can support integration cofactor function. Here, in experimentally useful human cell lines, we used TALENs to definitively eradicate LEDGF/p75 by deleting either all of PSIP1 or the exons that code for the integrase binding domain. HIV-1 replication was severely impaired in these PSIP1 knockout cells. Experiments in these cells also excluded a role for LEDGF/p75 in HIV-1 assembly and showed that the main ALLINI mechanism is LEDGF/p75 independent. Site-specific gene targeting of PSIP1 may have therapeutic potential for HIV-1 disease.
doi:10.1128/JVI.01397-14
PMCID: PMC4136317  PMID: 24942577
19.  Improvement in Student Science Proficiency Through InSciEd Out 
Zebrafish  2012;9(4):155-168.
Abstract
Integrated Science Education Outreach (InSciEd Out) is a collaboration formed between Mayo Clinic, Winona State University, and Rochester Public Schools (MN) with the shared vision of achieving excellence in science education. InSciEd Out employs an equitable partnership model between scientists, teachers, education researchers, and the community. Teams of teachers from all disciplines within a single school experience cutting-edge science using the zebrafish model system, as well as current pedagogical methods, during a summer internship at the Mayo Clinic. Within the internship, the teachers produce new curriculum that directly addresses opportunities for science education improvement at their own school. Zebrafish are introduced within the new curriculum to support a living model of the practice of science. Following partnership with the InSciEd Out program and 2 years of implementation in the classroom, teacher-interns from a K–8 public school reported access to local scientific technology and expertise they had not previously recognized. Teachers also reported improved integration of other disciplines into the scientific curriculum and a flow of concepts vertically from K through 8. Students more than doubled selection of an Honors science track in high school to nearly 90%. 98% of students who took the Minnesota Comprehensive Assessments in their 5th and 8th grade year (a span that includes 2 years of InSciEd Out) showed medium or high growth in science proficiency. These metrics indicate that cooperation between educators and scientists can result in positive change in student science proficiency and demonstrate that a higher expectation in science education can be achieved in US public schools.
doi:10.1089/zeb.2012.0818
PMCID: PMC3529492  PMID: 23244687
20.  A TALE of Two Nucleases: Gene Targeting for the Masses? 
Zebrafish  2011;8(3):147-149.
Abstract
Genome editing appears poised to enter an exciting new era. Targeted double-stranded breaks due to custom restriction enzymes are powerful nucleating events for the induction of local changes in the genome. The zinc finger nuclease (ZFN) platform established the potential of this approach for the zebrafish, but access to high quality reagents has been a major bottleneck for the field. However, two groups recently report successful somatic and germline gene modification using a new nuclease architecture, transcription activator-like effector nucleases (TALENs). TALEN construction is simpler, potentially more reliable, and in the few cases examined, shows fewer off-target effects than corresponding ZFNs. TALENs promise to bring gene targeting to the majority of zebrafish laboratories.
doi:10.1089/zeb.2011.9993
PMCID: PMC3174730  PMID: 21929364
21.  Making designer mutants in model organisms 
Development (Cambridge, England)  2014;141(21):4042-4054.
Recent advances in the targeted modification of complex eukaryotic genomes have unlocked a new era of genome engineering. From the pioneering work using zinc-finger nucleases (ZFNs), to the advent of the versatile and specific TALEN systems, and most recently the highly accessible CRISPR/Cas9 systems, we now possess an unprecedented ability to analyze developmental processes using sophisticated designer genetic tools. In this Review, we summarize the common approaches and applications of these still-evolving tools as they are being used in the most popular model developmental systems. Excitingly, these robust and simple genomic engineering tools also promise to revolutionize developmental studies using less well established experimental organisms.
doi:10.1242/dev.102186
PMCID: PMC4302887  PMID: 25336735
Genome engineering; Transcription activator-like effector nuclease (TALEN); Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas9); Zinc finger nuclease (ZFN); Model organisms
22.  SCORE Imaging: Specimen in a Corrected Optical Rotational Enclosure 
Zebrafish  2010;7(2):149-154.
Abstract
Visual data collection is paramount for the majority of scientific research. The added transparency of the zebrafish (Danio rerio) allows for a greater detail of complex biological research that accompanies seemingly simple observational tools. We developed a visual data analysis and collection approach that takes advantage of the cylindrical nature of the zebrafish allowing for an efficient and effective method for image capture that we call Specimen in a Corrected Optical Rotational Enclosure imaging. To achieve a nondistorted image, zebrafish were placed in a fluorinated ethylene propylene tube with a surrounding optically corrected imaging solution (water). By similarly matching the refractive index of the housing (fluorinated ethylene propylene tubing) to that of the inner liquid and outer liquid (water), distortion was markedly reduced, producing a crisp imagable specimen that is able to be fully rotated 360°. A similar procedure was established for fixed zebrafish embryos using convenient, readily available borosilicate capillaries surrounded by 75% glycerol. The method described here could be applied to chemical genetic screening and other related high-throughput methods within the fish community and among other scientific fields.
doi:10.1089/zeb.2010.0660
PMCID: PMC3117241  PMID: 20528262
23.  The Zebrafish as a Model to Study Polycystic Liver Disease 
Zebrafish  2013;10(2):211-217.
Abstract
In the polycystic liver diseases (PLD), genetic defects initiate the formation of cysts in the liver and kidney. In rodent models of PLD (i.e., the PCK rat and Pkd2WS25/− mouse), we have studied hepatorenal cystic disease and therapeutic approaches. In this study, we employed zebrafish injected with morpholinos against genes involved in the PLD, including sec63, prkcsh, and pkd1a. We calculated the liver cystic area, and based on our rodent studies, we exposed the embryos to pasireotide [1 μM] or vitamin K3 [100 μM] and assessed the endoplasmic reticulum (ER) in cholangiocytes in embryos treated with 4-phenylbutyrate (4-PBA). Our results show that (a) morpholinos against sec63, prkcsh, and pkd1a eliminate expression of the respective proteins; (b) phenotypic body changes included curved tail and the formation of hepatic cysts in zebrafish larvae; (c) exposure of embryos to pasireotide inhibited hepatic cystogenesis in the zebrafish models; and (d) exposure of embryos to 4-PBA resulted in the ER in cholangiocytes resolving from a curved to a smooth appearance. Our results suggest that the zebrafish model of PLD may provide a means to screen drugs that could inhibit hepatic cystogenesis.
doi:10.1089/zeb.2012.0825
PMCID: PMC3673589  PMID: 23668934
24.  Zinc Finger–Based Knockout Punches for Zebrafish Genes 
Zebrafish  2008;5(2):121-123.
The ability to manipulate the genome is critical to develop and test hypotheses based on genetics. Knockdown strategies focused on RNAi and/or morpholinos are excellent genetic tools, but they come with substantial technical limitations. A new gene targeting approach employing synthetic zinc finger nuclease (ZFN) technology is a powerful and complementary approach to directly modify genetic loci for many diverse applications, notably enhancing Danio rerio (the zebrafish) as an experimental organism for understanding human disease. This ZFN-based technology to generate targeted knockouts in this aquatic animal opens the door to an array of new biological models of human disease and genetic testing.
doi:10.1089/zeb.2008.9988
PMCID: PMC2849655  PMID: 18554175
25.  A Primer for Morpholino Use in Zebrafish 
Zebrafish  2009;6(1):69-77.
Morpholino oligonucleotides are the most common anti-sense “knockdown” technique used in zebrafish (Danio rerio). This review discusses common practices for the design, preparation, and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.
doi:10.1089/zeb.2008.0555
PMCID: PMC2776066  PMID: 19374550

Results 1-25 (63)