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1.  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.
PMCID: PMC3144740  PMID: 21746693
morpholinos; zebrafish; knockdown
2.  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.
PMCID: PMC3965580  PMID: 23948583
TALENs; zebrafish; iPS cells; genome engineering; cardiovascular disease modeling
3.  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.
PMCID: PMC4059519  PMID: 19918918
4.  A Sequence-Based Variation Map of Zebrafish 
Zebrafish  2013;10(1):15-20.
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.
PMCID: PMC3629779  PMID: 23590399
5.  The CRISPR System—Keeping Zebrafish Gene Targeting Fresh 
Zebrafish  2013;10(1):116-118.
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.
PMCID: PMC3629780  PMID: 23536990
6.  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.
PMCID: PMC2722259  PMID: 19109308
transposon; gene delivery; gene discovery; gene transfer vectors; vertebrates
7.  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.
PMCID: PMC4482254  PMID: 26110643
8.  Detection of 1α,25-Dihydroxyvitamin D-Regulated miRNAs in Zebrafish by Whole Transcriptome Sequencing 
Zebrafish  2014;11(3):207-218.
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.
PMCID: PMC4050706  PMID: 24650217
9.  Etv2 and Fli1b Function Together as Key Regulators of Vasculogenesis and Angiogenesis 
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.
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.
PMCID: PMC4427907  PMID: 25722433
angiogenesis; ETS transcription factor; vasculogenesis; zebrafish
10.  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.
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.
PMCID: PMC4136317  PMID: 24942577
11.  A TALE of Two Nucleases: Gene Targeting for the Masses? 
Zebrafish  2011;8(3):147-149.
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.
PMCID: PMC3174730  PMID: 21929364
12.  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.
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
13.  SCORE Imaging: Specimen in a Corrected Optical Rotational Enclosure 
Zebrafish  2010;7(2):149-154.
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.
PMCID: PMC3117241  PMID: 20528262
14.  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.
PMCID: PMC2849655  PMID: 18554175
15.  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.
PMCID: PMC2776066  PMID: 19374550
16.  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.
PMCID: PMC2776066  PMID: 19374550
17.  The Zebrafish as a Model to Study Polycystic Liver Disease 
Zebrafish  2013;10(2):211-217.
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.
PMCID: PMC3673589  PMID: 23668934
18.  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.
PMCID: PMC2849655  PMID: 18554175
19.  Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles 
Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.
PMCID: PMC3971575  PMID: 22361506
Myosin; Myomesin 3; M-line; Sarcomere
20.  Larval Zebrafish Model for FDA-Approved Drug Repositioning for Tobacco Dependence Treatment 
PLoS ONE  2014;9(3):e90467.
Cigarette smoking remains the most preventable cause of death and excess health care costs in the United States, and is a leading cause of death among alcoholics. Long-term tobacco abstinence rates are low, and pharmacotherapeutic options are limited. Repositioning medications approved by the U.S. Food and Drug Administration (FDA) may efficiently provide clinicians with new treatment options. We developed a drug-repositioning paradigm using larval zebrafish locomotion and established predictive clinical validity using FDA-approved smoking cessation therapeutics. We evaluated 39 physician-vetted medications for nicotine-induced locomotor activation blockade. We further evaluated candidate medications for altered ethanol response, as well as in combination with varenicline for nicotine-response attenuation. Six medications specifically inhibited the nicotine response. Among this set, apomorphine and topiramate blocked both nicotine and ethanol responses. Both positively interact with varenicline in the Bliss Independence test, indicating potential synergistic interactions suggesting these are candidates for translation into Phase II clinical trials for smoking cessation.
PMCID: PMC3962344  PMID: 24658307
21.  The Zebrafish GenomeWiki: a crowdsourcing approach to connect the long tail for zebrafish gene annotation 
A large repertoire of gene-centric data has been generated in the field of zebrafish biology. Although the bulk of these data are available in the public domain, most of them are not readily accessible or available in nonstandard formats. One major challenge is to unify and integrate these widely scattered data sources. We tested the hypothesis that active community participation could be a viable option to address this challenge. We present here our approach to create standards for assimilation and sharing of information and a system of open standards for database intercommunication. We have attempted to address this challenge by creating a community-centric solution for zebrafish gene annotation. The Zebrafish GenomeWiki is a ‘wiki’-based resource, which aims to provide an altruistic shared environment for collective annotation of the zebrafish genes. The Zebrafish GenomeWiki has features that enable users to comment, annotate, edit and rate this gene-centric information. The credits for contributions can be tracked through a transparent microattribution system. In contrast to other wikis, the Zebrafish GenomeWiki is a ‘structured wiki’ or rather a ‘semantic wiki’. The Zebrafish GenomeWiki implements a semantically linked data structure, which in the future would be amenable to semantic search.
Database URL:
PMCID: PMC3936183  PMID: 24578356
22.  Insertional mutagenesis strategies in zebrafish 
Genome Biology  2007;8(Suppl 1):S9.
We review here some recent developments in the field of insertional mutagenesis in zebrafish. We highlight the advantages and limitations of the rich body of retroviral methodologies, and we focus on the mechanisms and concepts of new transposon-based mutagenesis approaches under development, including prospects for conditional 'gene trapping' and 'gene breaking' approaches.
PMCID: PMC2106850  PMID: 18047701
23.  Trapping Cardiac Recessive Mutants via Expression-based Insertional Mutagenesis Screening 
Circulation research  2013;112(4):606-617.
Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ such as the heart, especially those exhibiting adult phenotypes upon depletion.
We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes.
Methods and Results
The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein (mRFP) reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of one embryonic lethal, one larval lethal, and one adult recessive mutant exhibiting cardiac hypertrophy at one year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, alpha a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy.
We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a Zebrafish Insertional Cardiac (ZIC) mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.
PMCID: PMC3603352  PMID: 23283723
Gene trapping; insertional mutagenesis screen; cardiac mutants; adult recessive; zebrafish; transposon
Gene expression patterns : GEP  2012;12(7-8):228-235.
Sclerostin is a highly conserved, secreted, cystine-knot protein which regulates osteoblast function. Humans with mutations in the sclerostin gene (SOST), manifest increased axial and appendicular skeletal bone density with attendant complications. In adult bone, sclerostin is expressed in osteocytes and osteoblasts. Danio rerio sclerostin-like protein is closely related to sea bass sclerostin, and is related to chicken and mammalian sclerostins. Little is known about the expression of sclerostin in early developing skeletal or extra-skeletal tissues. We assessed sclerostin (sost) gene expression in developing zebrafish (Danio rerio) embryos with whole mount is situ hybridization methods. The earliest expression of sost RNA was noted during 12 hours post-fertilization (hpf). At 15 hpf, sost RNA was detected in the developing nervous system and in Kupffer’s vesicle. At 18, 20 and 22 hpf, expression in rhombic lip precursors was seen. By 24 hpf, expression in the upper and lower rhombic lip and developing spinal cord was noted. Expression in the rhombic lip and spinal cord persisted through 28 hpf and then diminished in intensity through 44 hpf. At 28 hpf, sost expression was noted in developing pharyngeal cartilage; expression in pharyngeal cartilage increased with time. By 48 hpf, sost RNA was clearly detected in the developing pharyngeal arch cartilage. Sost RNA was abundantly expressed in the pharyngeal arch cartilage, and in developing pectoral fins, 72, 96 and 120 hpf. Our study is the first detailed analysis of sost gene expression in early metazoan development.
PMCID: PMC3435489  PMID: 22575304
Sclerostin; sost; skeleton; cartilage; brain
25.  High Efficiency In Vivo Genome Engineering with a Simplified 15-RVD GoldyTALEN Design 
PLoS ONE  2013;8(5):e65259.
Transcription activator-like effector nucleases (TALENs) enable genome engineering in cell culture and many organisms. Recently, the GoldyTALEN scaffold was shown to readily introduce mutations in zebrafish (Danio rerio) and livestock through non-homologous end joining (NHEJ) and homology-directed repair (HDR). To deploy the GoldyTALEN system for high-throughput mutagenesis in model organisms, a simple design with high efficacy is desirable. We tested the in vivo efficacy of a simplified 15-RVD GoldyTALEN design (spacer between 13–20 bp and T nucleotide preceding each TALEN binding site) in zebrafish. All 14 tested TALEN pairs (100%) introduced small insertions and deletions at somatic efficacy ranging from 24 to 86%, and mutations were inheritable at high frequencies (18–100%). By co-injecting two GoldyTALEN pairs, inheritable deletions of a large genomic fragment up to 18 kb were successfully introduced at two different loci. In conclusion, these high efficiency 15-RVD GoldyTALENs are useful for high-throughput mutagenesis in diverse application including hypothesis testing from basic science to precision medicine.
PMCID: PMC3667041  PMID: 23734242

Results 1-25 (55)