Craig J. Coates

piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty, Tol2, and Mos1 in mammalian cells

A nonviral vector for highly efficient site-specific integration would be desirable for many applications in transgenesis, including gene therapy. In this study we directly compared the genomic integration efficiencies of piggyBac, hyperactive Sleeping Beauty (SB11), Tol2, and Mos1 in four mammalian cell lines. piggyBac demonstrated significantly higher transposition activity in all cell lines whereas Mos1 had no activity. Furthermore, piggyBac transposase coupled to the GAL4 DNA-binding domain retains transposition activity whereas similarly manipulated gene products of Tol2 and SB11 were inactive. The high transposition activity of piggyBac and the flexibility for molecular modification of its transposase suggest the possibility of using it routinely for mammalian transgenesis.

Gene vector and transposable element behavior in mosquitoes

SUMMARY The development of efficient germ-line transformation technologies for mosquitoes has increased the ability of entomologists to find, isolate and analyze genes. The utility of the currently available systems will be determined by a number of factors including the behavior of the gene vectors during the initial integration event and their behavior after chromosomal integration. Post-integration behavior will determine whether the transposable elements being employed currently as primary gene vectors will be useful as gene-tagging and enhancer-trapping agents. The post-integration behavior of existing insect vectors has not been extensively examined. Mos1 is useful as a primary germ-line transformation vector in insects but is inefficiently remobilized in Drosophila melanogaster and Aedes aegypti. Hermes transforms D. melanogaster efficiently and can be remobilized in this species. This element is also useful for creating transgenic A. aegypti, but its mode of integration in mosquitoes results in the insertion of flanking plasmid DNA. Hermes can be remobilized in the soma of A. aegypti and transposes using a common cut-and-paste mechanism; however, the element does not remobilize in the germ line. piggyBac can be used to create transgenic mosquitoes and occasionally integrates using a mechanism other than a simple cut-and-paste mechanism. Preliminary data suggest that remobilization is infrequent. Minos also functions in mosquitoes and, like the other gene vectors, appears to remobilize inefficiently following integration. These results have implications for future gene vector development efforts and applications.

The Hermes element from Musca domestica can transpose in four families of cyclorrhaphan flies

Transgenic insect technology will provide opportunities to explore the basic biology of a broad range of insect species in ways that will prove insightful and important. It is also a technology that will provide opportunities to manipulate the genotypes of insects of practical significance to the health and welfare of humans. The Hermes transposable element from the housefly, Musca domestica, is a short inverted repeat-type element related to hobo from Drosophila melanogaster, Ac from Zea mays, and Tam3 from Antirrhinum majus. It has potential to become a versatile and efficient broad host-range insect transformation vector. The ability of Hermes to transpose when introduced into five species of diptera from four divergent families was tested using an in vivo, interplasmid transpositional recombination assay. Hermes was capable of transposing in all species tested, demonstrating that Hermes has a broad host-range. In addition, the rates of transposition were sufficiently high in all species tested to suggest that Hermes will be an efficient gene transfer vector in a wide range of insect species. The Hermes element also revealed a pattern of integration into the target substrate that permitted factors determining integration site selection to be identified. Primary nucleotide sequence of the integration site played a role as did proximity to preferred integration sites and the nucleosomal organization of the target.

Chimeric Mos1 and piggyBac transposases result in site-directed integration

Genetic transformation systems based on Mos1 and piggyBac transposable elements are used to achieve stable chromosomal integration. However, integration sites are randomly distributed in the genome and transgene expression can be influenced by position effects. We developed a novel technology that utilizes chimeric transposases to direct integration into specific sites on a target DNA molecule. The Gal4 DNA binding domain was fused to the NH2 terminus of the Mos1 and piggyBac transposases and a target plasmid was created that contained upstream activating sequences (UAS), to which the Gal4 DBD binds with high affinity. The transpositional activity of the Gal4‐Mos1 transposase was 12.7‐fold higher compared to controls where the Gal4‐UAS interaction was absent and 96% of the recovered transposition products were identical, with integration occurring at the same TA site. In a parallel experiment, a Gal4‐piggyBac transposase resulted in an 11.6‐fold increase in transpositional activity compared to controls, with 67% of the integrations occurring at a single TTAA site. This technology has the potential to minimize nonspecific integration events that may result in insertional mutagenesis and reduced fitness. Site‐directed integration will be advantageous to the manipulation of genomes, study of gene function, and for the development of gene therapy techniques.—Maragathavally, K. J., Kaminski, J. M., Coates, C. J. Chimeric Mos1 and piggyBac transposases result in site‐directed integration. FASEB J. 20, E1188‐E1195 (2006)

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

Structure of Hermes integrations in the germline of the yellow fever mosquito, Aedes aegypti

The Hermes transposable element is derived from the house fly, Musca domestica, and can incorporate into the germline of the yellow fever mosquito, Aedes aegypti. Preliminary Southern analyses indicated that Hermes integrated along with the marker gene into the mosquito genomic DNA. Here we show that Hermes integrations are accompanied by the integration of the donor plasmid as well. In addition, breaks in the donor plasmid DNAs do not occur precisely, or at the end of the terminal inverted repeats, and are accompanied by small deletions in the plasmids. Furthermore, integrations do not cause the typical 8‐bp duplications of the target site DNA. No integrations are observed in the absence of a source of Hermes transposase. The Hermes transposase clearly did not catalyse precise cut‐and‐paste transposition in these transformed lines. It may have integrated the transposon through general recombination or through a partial replicative transposition mechanism. The imprecision of Hermes integration may result from interactions of the transposase with an endogenous hAT‐like element in the mosquito genome.

Active integration: new strategies for transgenesis

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed “Active Transgenesis” to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac’s specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti.

The Class II transposable element, piggyBac, was used to transform the yellow fever mosquito, Aedes aegypti. In two transformed lines only 15–30 of progeny inherited the transgene, with these individuals displaying mosaic expression of the EGFP marker gene. Southern analyses, gene amplification of genomic DNA, and plasmid rescue experiments provided evidence that these lines contained a high copy number of piggyBac transformation constructs and that much of this DNA consisted of both donor and helper plasmids. A detailed analysis of one line showed that the majority of piggyBac sequences were unit-length donor or helper plasmids arranged in a large tandem array that could be lost en masse in a single generation. Despite the presence of a transposase source and many intact donor elements, no conservative (cut and paste) transposition of piggyBac was observed in these lines. These results reveal one possible outcome of uncontrolled and/or unexpected recombination in this mosquito, and support the conclusion that further investigation is necessary before transposable elements such as piggyBac can be used as genetic drive mechanisms to move pathogen-resistance genes into mosquito populations.

Detection of differences in oligonucleotide-influenced aggregation of colloidal gold nanoparticles using absorption spectroscopy

A rapid, simple, and reproducible assay is described that can be used to detect differences in the ability of oligonucleotides to influence the aggregation of colloidal gold nanoparticles. The aggregation reaction of the gold colloid was monitored through UV-visible absorption spectroscopy. Single isolated colloidal gold particles have a surface plasmon resonance manifested as a single absorbance peak at approximately 520 nm, and aggregated gold complexes develop new red-shifted peaks/shoulders depending on the nature and extent of the aggregated complex. A simple ratiometric study of the area under the single and aggregated plasmon resonance peaks thus gives information about the extent of the aggregation. It is postulated that differences in dynamic flexibility of the oligonucleotides affect their influence on the aggregation state of the gold nanoparticles. The results of this study provide new clues toward unraveling the causes behind the preferential affinity of the Hermes transposable element for certain insertion sites compared to other sequences that also contain recognizable target sites. The technique is robust and thus can potentially be used to study similar questions for numerous transposable elements and target sequences.

High-level gene expression in Aedes albopictus cells using a baculovirus Hr3 enhancer and IE1 transactivator

BackgroundAedes aegypti is the key vector of both the Yellow Fever and Dengue Fever viruses throughout many parts of the world. Low and variable transgene expression levels due to position effect and position effect variegation are problematic to efforts to create transgenic laboratory strains refractory to these viruses. Transformation efficiencies are also less than optimal, likely due to failure to detect expression from all integrated transgenes and potentially due to limited expression of the transposase required for transgene integration.ResultsExpression plasmids utilizing three heterologous promoters and three heterologous enhancers, in all possible combinations, were tested. The Hr3/IE1 enhancer-trans activator in combination with each of the constitutive heterologous promoters tested increased reporter gene expression significantly in transiently transfected Aedes albopictus C7-10 cells.ConclusionsThe addition of the Hr3 enhancer to expression cassettes and concomitant expression of the IE1 trans activator gene product is a potential method for increasing the level of transgene expression in insect systems. This mechanism could also potentially be used to increase the level of transiently-expressed transposase in order to increase the number of integration events in transposon-mediated transformation experiments.