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1.  Genome edited sheep and cattle 
Transgenic Research  2014;24:147-153.
Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.
PMCID: PMC4274373  PMID: 25204701
Livestock; TALEN; Myostatin; Zygote; Genetic engineering
2.  Simple and Efficient Methods for Enrichment and Isolation of Endonuclease Modified Cells 
PLoS ONE  2014;9(5):e96114.
The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.
PMCID: PMC4010432  PMID: 24798371
3.  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.
PMCID: PMC3010673  PMID: 21197395
4.  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
5.  Precision Editing of Large Animal Genomes 
Advances in genetics  2012;80:37-97.
Transgenic animals are an important source of protein and nutrition for most humans and will play key roles in satisfying the increasing demand for food in an ever-increasing world population. The past decade has experienced a revolution in the development of methods that permit the introduction of specific alterations to complex genomes. This precision will enhance genome-based improvement of farm animals for food production. Precision genetics also will enhance the development of therapeutic biomaterials and models of human disease as resources for the development of advanced patient therapies.
PMCID: PMC3683964  PMID: 23084873
6.  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.
PMCID: PMC3491146  PMID: 23000899
zebrafish; TALEN; genome engineering; loxP
7.  Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition 
Transgenic research  2010;20(1):29-45.
Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.
PMCID: PMC3516389  PMID: 20352328
Sleeping Beauty; Transposon; Transgenesis; Mouse; Rat; Methylation
8.  Targeting DNA With Fingers and TALENs 
PMCID: PMC3381595  PMID: 23344620
9.  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.
PMCID: PMC2651795  PMID: 19136468
10.  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).
PMCID: PMC2106845  PMID: 18047690
11.  The Restriction of Zoonotic PERV Transmission by Human APOBEC3G 
PLoS ONE  2007;2(9):e893.
The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections.
PMCID: PMC1963317  PMID: 17849022
12.  Enzymatic engineering of the porcine genome with transposons and recombinases 
BMC Biotechnology  2007;7:42.
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.
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.
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.
PMCID: PMC1939997  PMID: 17640337
13.  Evolutionarily conserved and non-conserved retrovirus restriction activities of artiodactyl APOBEC3F proteins 
Nucleic Acids Research  2006;34(19):5683-5694.
The APOBEC3 proteins are unique to mammals. Many inhibit retrovirus infection through a cDNA cytosine deamination mechanism. HIV-1 neutralizes this host defense through Vif, which triggers APOBEC3 ubiquitination and degradation. Here, we report an APOBEC3F-like, double deaminase domain protein from three artiodactyls: cattle, pigs and sheep. Like their human counterparts, APOBEC3F and APOBEC3G, the artiodactyl APOBEC3F proteins are DNA cytosine deaminases that locate predominantly to the cytosol and can inhibit the replication of HIV-1 and MLV. Retrovirus restriction is attributable to deaminase-dependent and -independent mechanisms, as deaminase-defective mutants retain significant anti-retroviral activity. However, unlike human APOBEC3F and APOBEC3G, the artiodactyl APOBEC3F proteins have an active N-terminal DNA cytosine deaminase domain, which elicits a broader dinucleotide deamination preference, and they are resistant to HIV-1 Vif. These data indicate that DNA cytosine deamination; sub-cellular localization and retrovirus restriction activities are conserved in mammals, whereas active site location, local mutational preferences and Vif susceptibility are not. Together, these studies indicate that some properties of the mammal-specific, APOBEC3-dependent retroelement restriction system are necessary and conserved, but others are simultaneously modular and highly adaptable.
PMCID: PMC1636497  PMID: 17038330
14.  Identifying secretomes in people, pufferfish and pigs 
Nucleic Acids Research  2004;32(4):1414-1421.
The proteins processed by the secretory pathway (secretome) are critical players in the development of multi-cellular eukaryotic organisms but have yet to be comprehensively studied at the genomic level. In this study, we use the Target P algorithm to predict human (13–20% of proteins found in individual datasets) and Fugu (14%) secretomes based on analysis of their nearly complete proteomes. We combine internal processing with prediction software to automate secreted protein identification and overcome one of the major challenges associated with EST data: identification of the minority of clones that encode N-terminally-complete proteins. We discuss the use of these methods to predict secreted proteins in EST-based consensus sequence sets, and we validate these predictions using an assay for cell-free cotranslational translocation. Analysis of TIGR Porcine Gene Index 4.0 as a test dataset resulted in the identification of 352 N-terminally-complete, putative secreted proteins. In functional agreement with our predictions, 34 of 40 (85%) of these cDNAs were verified to be cotranslationally translocated in an in vitro translation system. The methods developed here are specifically designed to accept partial open reading frames and improve secreted protein predictions in eukaryotic transcriptomes, and are valuable for the analysis and annotation of eukaryotic EST databases.
PMCID: PMC390277  PMID: 14990746
15.  Comparison of computational methods for identifying translation initiation sites in EST data 
BMC Bioinformatics  2004;5:14.
Expressed Sequence Tag (EST) sequences are generally single-strand, single-pass sequences, only 200–600 nucleotides long, contain errors resulting in frame shifts, and represent different parts of their parent cDNA. If the cDNAs contain translation initiation sites, they may be suitable for functional genomics studies. We have compared five methods to predict translation initiation sites in EST data: first-ATG, ESTScan, Diogenes, Netstart, and ATGpr.
A dataset of 100 EST sequences, 50 with and 50 without, translation initiation sites, was created. Based on analysis of this dataset, ATGpr is found to be the most accurate for predicting the presence versus absence of translation initiation sites. With a maximum accuracy of 76%, ATGpr more accurately predicts the position or absence of translation initiation sites than NetStart (57%) or Diogenes (50%). ATGpr similarly excels when start sites are known to be present (90%), whereas NetStart achieves only 60% overall accuracy. As a baseline for comparison, choosing the first ATG correctly identifies the translation initiation site in 74% of the sequences. ESTScan and Diogenes, consistent with their intended use, are able to identify open reading frames, but are unable to determine the precise position of translation initiation sites.
ATGpr demonstrates high sensitivity, specificity, and overall accuracy in identifying start sites while also rejecting incomplete sequences. A database of EST sequences suitable for validating programs for translation initiation site prediction is now available. These tools and materials may open an avenue for future improvements in start site prediction and EST analysis.
PMCID: PMC375524  PMID: 15053846

Results 1-15 (15)