PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (48)
 

Clipboard (0)
None

Select a Filter Below

Year of Publication
more »
1.  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
2.  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.
doi:10.1016/j.jgg.2012.01.005
PMCID: PMC3971575  PMID: 22361506
Myosin; Myomesin 3; M-line; Sarcomere
3.  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
4.  Trapping Cardiac Recessive Mutants via Expression-based Insertional Mutagenesis Screening 
Circulation research  2013;112(4):606-617.
Rationale
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.
Objective
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.
Conclusions
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.
doi:10.1161/CIRCRESAHA.112.300603
PMCID: PMC3603352  PMID: 23283723
Gene trapping; insertional mutagenesis screen; cardiac mutants; adult recessive; zebrafish; transposon
5.  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
6.  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
7.  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
8.  EXPRESSION OF SCLEROSTIN IN THE DEVELOPING ZEBRAFISH (DANIO) BRAIN AND SKELETON 
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.
doi:10.1016/j.gep.2012.04.003
PMCID: PMC3435489  PMID: 22575304
Sclerostin; sost; skeleton; cartilage; brain
9.  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.
doi:10.1371/journal.pone.0065259
PMCID: PMC3667041  PMID: 23734242
10.  In vivo Genome Editing Using High Efficiency TALENs 
Nature  2012;491(7422):114-118.
The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease using a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications1–5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show this enhanced TALEN toolkit demonstrates a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino (MO)-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA (ssDNA) oligonucleotides (oligos) to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair (HDR), including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.
doi:10.1038/nature11537
PMCID: PMC3491146  PMID: 23000899
zebrafish; TALEN; genome engineering; loxP
11.  Predictors of indoor absolute humidity and estimated effects on influenza virus survival in grade schools 
Background
Low absolute humidity (AH) has been associated with increased influenza virus survival and transmissibility and the onset of seasonal influenza outbreaks. Humidification of indoor environments may mitigate viral transmission and may be an important control strategy, particularly in schools where viral transmission is common and contributes to the spread of influenza in communities. However, the variability and predictors of AH in the indoor school environment and the feasibility of classroom humidification to levels that could decrease viral survival have not been studied.
Methods
Automated sensors were used to measure temperature, humidity and CO2 levels in two Minnesota grade schools without central humidification during two successive winters. Outdoor AH measurements were derived from the North American Land Data Assimilation System. Variability in indoor AH within classrooms, between classrooms in the same school, and between schools was assessed using concordance correlation coefficients (CCC). Predictors of indoor AH were examined using time-series Auto-Regressive Conditional Heteroskedasticity models. Classroom humidifiers were used when school was not in session to assess the feasibility of increasing indoor AH to levels associated with decreased influenza virus survival, as projected from previously published animal experiments.
Results
AH varied little within classrooms (CCC >0.90) but was more variable between classrooms in the same school (CCC 0.81 for School 1, 0.88 for School 2) and between schools (CCC 0.81). Indoor AH varied widely during the winter (range 2.60 to 10.34 millibars [mb]) and was strongly associated with changes in outdoor AH (p < 0.001). Changes in indoor AH on school weekdays were strongly associated with CO2 levels (p < 0.001). Over 4 hours, classroom humidifiers increased indoor AH by 4 mb, an increase sufficient to decrease projected 1-hour virus survival by an absolute value of 30% during winter months.
Conclusions
During winter, indoor AH in non-humidified grade schools varies substantially and often to levels that are very low. Indoor results are predicted by outdoor AH over a season and CO2 levels (which likely reflects human activity) during individual school days. Classroom humidification may be a feasible approach to increase indoor AH to levels that may decrease influenza virus survival and transmission.
doi:10.1186/1471-2334-13-71
PMCID: PMC3568414  PMID: 23383620
Influenza; Humidity; Schools; Climate
12.  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.
doi:10.1002/dvdy.23725
PMCID: PMC3448447  PMID: 22275008
Tol2 gene trap; endothelial cells; vein; vasculature; central nervous system
13.  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
14.  Mojo Hand, a TALEN design tool for genome editing applications 
BMC Bioinformatics  2013;14:1.
Background
Recent studies of transcription activator-like (TAL) effector domains fused to nucleases (TALENs) demonstrate enormous potential for genome editing. Effective design of TALENs requires a combination of selecting appropriate genetic features, finding pairs of binding sites based on a consensus sequence, and, in some cases, identifying endogenous restriction sites for downstream molecular genetic applications.
Results
We present the web-based program Mojo Hand for designing TAL and TALEN constructs for genome editing applications (http://www.talendesign.org). We describe the algorithm and its implementation. The features of Mojo Hand include (1) automatic download of genomic data from the National Center for Biotechnology Information, (2) analysis of any DNA sequence to reveal pairs of binding sites based on a user-defined template, (3) selection of restriction-enzyme recognition sites in the spacer between the TAL monomer binding sites including options for the selection of restriction enzyme suppliers, and (4) output files designed for subsequent TALEN construction using the Golden Gate assembly method.
Conclusions
Mojo Hand enables the rapid identification of TAL binding sites for use in TALEN design. The assembly of TALEN constructs, is also simplified by using the TAL-site prediction program in conjunction with a spreadsheet management aid of reagent concentrations and TALEN formulation. Mojo Hand enables scientists to more rapidly deploy TALENs for genome editing applications.
doi:10.1186/1471-2105-14-1
PMCID: PMC3575288  PMID: 23323762
TAL effector; TALEN; Genome editing
15.  Zinc Finger–Based Knockout Punches for Zebrafish Genes 
Zebrafish  2008;5(2):121-123.
Abstract
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
16.  Stressing Zebrafish for Behavioral Genetics 
Reviews in the neurosciences  2011;22(1):49-62.
Synopsis
The stress response is a normal reaction to a real or perceived threat. However, stress response systems that are overwhelmed or out of balance can increase both the incidence and severity of diseases including addiction and mood and anxiety disorders. Using an animal model with both genetic diversity and large family size can help discover the specific genetic and environmental contributions to these behavioral diseases. The stress response has been studied extensively in teleosts because of their importance in food production. The zebrafish (Danio rerio) is a major model organism with a strong record for use in developmental biology, genetic screening, and genomic studies. More recently, the stress response of larval and adult zebrafish has been documented. High-throughput automated tracking systems make possible behavioral readouts of the stress response in zebrafish. This non-invasive measure of the stress response can be combined with mutagenesis methods to dissect the genes involved in complex stress response behaviors in vertebrates. Understanding the genetic and epigenetic basis for the stress response in vertebrates will help to develop advanced screening and therapies for stress-aggravated diseases like addiction and mood and anxiety disorders.
doi:10.1515/RNS.2011.007
PMCID: PMC3470424  PMID: 21615261
17.  Transgenic Zebrafish Using Transposable Elements 
Methods in cell biology  2011;104:137-149.
DNA transposons are effective chromosomal engineering vehicles for making transgenic zebrafish. We describe both autonomous and non-autonomous transposable elements, and we compare and contrast popular transposon systems. The Tol2 system is a robust gene transfer tool and has been selected as the primary transposon platform, facilitating the development of an array of reagents readily shared within the zebrafish community. We present common transposon and transposase vectors within the field based on the Tol2 system. We describe methods with a high success rate of generating transgenic zebrafish using Tol2 vectors, including key quality control steps during the transgenesis process. Together, this data should enable the ready generation of transgenic zebrafish for scientific inquiry.
doi:10.1016/B978-0-12-374814-0.00008-2
PMCID: PMC3454445  PMID: 21924161
18.  An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish 
PLoS ONE  2012;7(9):e45240.
Background
Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo.
Methods and Results
Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth.
Conclusion
Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.
doi:10.1371/journal.pone.0045240
PMCID: PMC3441694  PMID: 23028871
19.  Zebrafish for the Study of the Biological Effects of Nicotine 
Nicotine & Tobacco Research  2011;13(5):301-312.
Introduction:
Zebrafish are emerging as a powerful animal model for studying the molecular and physiological effects of nicotine exposure. The zebrafish have many advantageous physical characteristics, including small size, high fecundity rates, and externally developing transparent embryos. When combined with a battery of molecular–genetic tools and behavioral assays, these attributes enable studies to be conducted that are not practical using traditional animal models.
Methods:
We reviewed the literature on the application of the zebrafish model as a preclinical model to study the biological effects of nicotine exposure.
Results:
The identified studies used zebrafish to examine the effects of nicotine exposure on early development, addiction, anxiety, and learning. The methods used included green fluorescent protein–labeled proteins to track in vivo nicotine-altered neuron development, nicotine-conditioned place preference, and locomotive sensitization linked with high-throughput molecular and genetic screens and behavioral models of learning and stress response to nicotine. Data are presented on the complete homology of all known human neural nicotinic acetylcholine receptors in zebrafish and on the biological similarity of human and zebrafish dopaminergic signaling.
Conclusions:
Tobacco dependence remains a major health problem worldwide. Further understanding of the molecular effects of nicotine exposure and genetic contributions to dependence may lead to improvement in patient treatment strategies. While there are limitations to the use of zebrafish as a preclinical model, it should provide a valuable tool to complement existing model systems. The reviewed studies demonstrate the enormous opportunity zebrafish have to advance the science of nicotine and tobacco research.
doi:10.1093/ntr/ntr010
PMCID: PMC3145391  PMID: 21385906
20.  in vivo protein trapping produces a functional expression codex of the vertebrate proteome 
Nature Methods  2011;8(6):506-515.
We describe a conditional in vivo protein trap mutagenesis system that reveals spatio-temporal protein expression dynamics and assesses gene function in the vertebrate Danio rerio. Integration of pGBT-RP2 (RP2), a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with >97% knockdown of normal transcript levels while simultaneously reporting protein expression of each locus. The mutant alleles are revertible in somatic tissues via Cre recombinase or splice-site blocking morpholinos, thus representing the first systematic conditional mutant alleles outside the mouse model. We report a collection of 350 zebrafish lines including a diverse array of 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 towards a unified codex of protein expression and direct functional annotation of the vertebrate genome.
doi:10.1038/nmeth.1606
PMCID: PMC3306164  PMID: 21552255
21.  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.
doi:10.1371/journal.pone.0090467
PMCID: PMC3962344  PMID: 24658307
22.  Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line 
Experimental hematology  2010;39(1):114-123.e1-5.
Objective
The microenvironment wherein hematopoietic stem cells (HSC) reside orchestrates HSC self-renewal vs. differentiation decisions. Stromal cells derived from ontogenically divergent hematopoietic microenvironments can support HSC in vitro and have been used to decipher factors that influence HSC fate decisions. Employing stromal cell lines derived from the AGM and embryonic liver, we aim to identify secreted factors that maintain/expand HSC in vitro.
Materials and Methods
We cultured murine lineage antigen negative (Lin−) bone marrow (BM) cells in transwells above the UG26-1B6, urogenitial ridge-, and EL08-1D2, embryonic liver-, derived cell lines. We, also, performed RT-qPCR analysis to identify differentially expressed genes from the Wnt family of proteins in ontogenically different stromal cell lines.
Results
Lin− murine BM cells maintained for 3 weeks in transwells above UG26-1B6 but not EL08-1D2 cells contained competitive repopulating HSC. Addition of as few as 25% UG26-1B6 cells to EL08-1D2 feeders led to maintenance of HSC in non-contact cultures, validating soluble factors are secreted by the UG26-1B6 cells. As we found that Wnt5a was significantly higher expressed in UG26-1B6 than EL08-1D2 cells, we added Wnt5a to EL08-1D2 transwell cultures or an antibody against Wnt5a to UG26-1B6 transwell cultures. Addition of Wnt5a to EL08-1D2 transwell cultures restored maintenance of HSC, whereas addition of an anti-Wnt5a antibody to UG26-1B6 transwell cultures inhibited maintenance of CR-HSC.
Conclusions
We demonstrate that stromal cell lines generated from embryonic microenvironments provide a tool to identify secreted proteins that play a role in the maintenance of HSC, and that at least one of the factors produced by UG26-1B6 cells, responsible for preserving HSC, is Wnt5a.
doi:10.1016/j.exphem.2010.09.010
PMCID: PMC3031906  PMID: 20933051
23.  zfishbook: connecting you to a world of zebrafish revertible mutants 
Nucleic Acids Research  2011;40(D1):D907-D911.
zfishbook is an internet-based openly accessible database of revertible protein trap gene-breaking transposon (GBT) insertional mutants in the zebrafish, Danio rerio. In these lines, a monomeric red fluorescent protein (mRFP) is encoded by an artificial 3′ exon, resulting in a translational fusion to endogenous loci. The natural transparency of the zebrafish embryo and larvae greatly facilitates the expression annotation of tagged loci using new capillary-based SCORE imaging methods. Molecular annotation of each line is facilitated by cloning methods such as 5′-Rapid Amplification of cDNA Ends (RACE) and inverse polymerase chain reaction (PCR). zfishbook (http://zfishbook.org) represents a central hub for molecular, expression and mutational information about GBT lines from the International Zebrafish Protein Trap Consortium (IZPTC) that includes researchers from around the globe. zfishbook is open to community-wide contributions including expression and functional annotation. zfishbook also represents a central location for information on how to obtain these lines from diverse members of the IZPTC and integration within other zebrafish community databases including Zebrafish Information Network (ZFIN), Ensembl and National Center for Biotechnology Information.
doi:10.1093/nar/gkr957
PMCID: PMC3245101  PMID: 22067444
24.  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: http://genome.igib.res.in/twiki
doi:10.1093/database/bau011
PMCID: PMC3936183  PMID: 24578356
25.  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

Results 1-25 (48)