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1.  Combination of Reverse and Chemical Genetic Screens Reveals Angiogenesis Inhibitors and Targets 
Chemistry & biology  2009;16(4):432-441.
SUMMARY
We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.
doi:10.1016/j.chembiol.2009.02.010
PMCID: PMC3984492  PMID: 19389629
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.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  Development and Application of Bovine and Porcine Oligonucleotide Arrays with Protein-Based Annotation 
The design of oligonucleotide sequences for the detection of gene expression in species with disparate volumes of genome and EST sequence information has been broadly studied. However, a congruous strategy has yet to emerge to allow the design of sensitive and specific gene expression detection probes. This study explores the use of a phylogenomic approach to align transcribed sequences to vertebrate protein sequences for the detection of gene families to design genomewide 70-mer oligonucleotide probe sequences for bovine and porcine. The bovine array contains 23,580 probes that target the transcripts of 16,341 genes, about 72% of the total number of bovine genes. The porcine array contains 19,980 probes targeting 15,204 genes, about 76% of the genes in the Ensembl annotation of the pig genome. An initial experiment using the bovine array demonstrates the specificity and sensitivity of the array.
doi:10.1155/2010/453638
PMCID: PMC3010673  PMID: 21197395
19.  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
20.  A Primer for Morpholino Use in Zebrafish 
Zebrafish  2009;6(1):69-77.
Abstract
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
21.  Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells 
Nucleic Acids Research  2009;37(4):1239-1247.
The Tc1/mariner family of DNA transposons is widespread across fungal, plant and animal kingdoms, and thought to contribute to the evolution of their host genomes. To date, an active Tc1 transposon has not been identified within the native genome of a vertebrate. We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells. In transposition assays, we found that the Passport transposon system improved stable cellular transgenesis by 40-fold, has an apparent preference for insertion into genes, and is subject to overproduction inhibition like other Tc1 elements. Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active. The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements.
doi:10.1093/nar/gkn1025
PMCID: PMC2651795  PMID: 19136468
22.  Pigs taking wing with transposons and recombinases 
Genome Biology  2007;8(Suppl 1):S13.
Swine production has been an important part of our lives since the late Mesolithic or early Neolithic periods, and ranks number one in world meat production. Pig production also contributes to high-value-added medical markets in the form of pharmaceuticals, heart valves, and surgical materials. Genetic engineering, including the addition of exogenous genetic material or manipulation of the endogenous genome, holds great promise for changing pig phenotypes for agricultural and medical applications. Although the first transgenic pigs were described in 1985, poor survival of manipulated embryos; inefficiencies in the integration, transmission, and expression of transgenes; and expensive husbandry costs have impeded the widespread application of pig genetic engineering. Sequencing of the pig genome and advances in reproductive technologies have rejuvenated efforts to apply transgenesis to swine. Pigs provide a compelling new resource for the directed production of pharmaceutical proteins and the provision of cells, vascular grafts, and organs for xenotransplantation. Additionally, given remarkable similarities in the physiology and size of people and pigs, swine will increasingly provide large animal models of human disease where rodent models are insufficient. We review the challenges facing pig transgenesis and discuss the utility of transposases and recombinases for enhancing the success and sophistication of pig genetic engineering. 'The paradise of my fancy is one where pigs have wings.' (GK Chesterton).
doi:10.1186/gb-2007-8-s1-s13
PMCID: PMC2106845  PMID: 18047690
23.  Enzymatic engineering of the porcine genome with transposons and recombinases 
BMC Biotechnology  2007;7:42.
Background
Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs.
Results
Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons.
Conclusion
We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.
doi:10.1186/1472-6750-7-42
PMCID: PMC1939997  PMID: 17640337
24.  Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon 
BMC Biotechnology  2006;6:30.
Background
Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern.
Results
Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA) system that is capable of activating the expression of genes under control of a Tet response element (TRE) promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns.
Conclusion
Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene-trap tTA could provide a means for both annotating mouse genes and creating a resource of mice that express a regulable transcription factor in temporally- and tissue-specific patterns for conditional gene expression studies. These mice would be a valuable resource to the mouse genetics community for purpose of dissecting mammalian gene function.
doi:10.1186/1472-6750-6-30
PMCID: PMC1557845  PMID: 16800892

Results 1-24 (24)