Ernst A. Wimmer

Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite

Malaria is estimated to cause 0.7 to 2.7 million deaths per year, but the actual figures could be substantially higher owing to under-reporting and difficulties in diagnosis. If no new control measures are developed, the malaria death toll is projected to double in the next 20 years. Efforts to control the disease are hampered by drug resistance in the Plasmodium parasites, insecticide resistance in mosquitoes, and the lack of an effective vaccine. Because mosquitoes are obligatory vectors for malaria transmission, the spread of malaria could be curtailed by rendering them incapable of transmitting parasites. Many of the tools required for the genetic manipulation of mosquito competence for malaria transmission have been developed. Foreign genes can now be introduced into the germ line of both culicine and anopheline mosquitoes, and these transgenes can be expressed in a tissue-specific manner. Here we report on the use of such tools to generate transgenic mosquitoes that express antiparasitic genes in their midgut epithelium, thus rendering them inefficient vectors for the disease. These findings have significant implications for the development of new strategies for malaria control.

A versatile vector set for animal transgenesis.

Abstract Genetic manipulation of a series of diverged arthropods is a highly desirable goal for a better understanding of developmental and evolutionary processes. A major obstacle so far has been the difficulty in obtaining marker genes that allow easy and reliable identification of transgenic animals. Here, we present a versatile vector set for germline transformation based on the promiscuous transposons mariner, Hermes and piggyBac. Into these vectors, we introduced a potentially universal marker system that is comprised of an artificial promoter containing three Pax-6 homodimer binding sites. This promoter drives strong expression of spectral variants of the enhanced green fluorescent protein (EGFP) in larval, pupal, and adult photoreceptors. Using special filter sets, the yellow (EYFP) and cyan (ECFP) variant are fully distinguishable and therefore represent a separable pair of markers. Furthermore, we adapted a simple plasmid-based transposition assay system to enable quick functional tests of our vectors in different arthropod species before employing them in more laborious germline transformation experiments. Using this system we demonstrate that our vectors transpose in both Drosophila melanogaster and Drosophila virilis.

A universal marker for transgenic insects.

Genetic manipulation of insects and other arthropods may enable better control strategies to be developed against agricultural pests and disease vectors. Transposon-based transformation techniques have been implemented in Drosophila and other insects such as medflies and mosquitoes. A major obstacle in the use of these transposons, however, has been the difficulty in obtaining marker genes that will allow easy and reliable identification of transgenic animals. Here we describe a marker system that is suitable for following gene transfer in most arthropods and in many other phyla.

Genetic techniques: A universal marker for transgenic insects

Genetic manipulation of insects and other arthropods may enable better control strategies to be developed against agricultural pests and disease vectors. Transposon-based transformation techniques have been implemented in Drosophila and other insects such as medflies and mosquitoes. A major obstacle in the use of these transposons, however, has been the difficulty in obtaining marker genes that will allow easy and reliable identification of transgenic animals. Here we describe a marker system that is suitable for following gene transfer in most arthropods and in many other phyla.

Highly sensitive, fluorescent transformation marker for Drosophila transgenesis

Abstract The efficiency of transposon-mediated germline transformation is dependent on the transposon mobility in the host embryo, and on the detectability of the used transformation marker. Therefore, high susceptibility of the transformation marker to position effect suppression is a disadvantage. Here we present data that the eye-specific expression of green fluorescent protein, driven by the 3xP3-EGFP marker, outperforms the commonly used ”mini”-white transformation marker in Drosophila germline transformation experiments: 3xP3-EGFP is more sensitive than ”mini”-white in identifying transgenic individuals and reacts differently to position effect suppression. Therefore, 3xP3-EGFP offers an ideal marker for applications in functional genomics where as many gene loci as possible should be targeted in the genome of a specific organism, for example, as intended in the Drosophila gene disruption project. Furthermore, we give a detailed description of the embryonic and larval expression mediated by the 3xP3-EGFP marker. These pre-adult expression patterns, and the potentially universal applicability of the transformation marker also offer additional advantages for selecting transgenic individuals in organisms other than Drosophila. This will be of great interest to the field of evolutionary developmental biology and to modern pest management programs.

Fluorescent transformation markers for insect transgenesis.

The first effectively achieved germ-line transformations of non-drosophilid insects were based on mutant rescue of eye color phenotypes. However, for most insect species neither visible mutants nor corresponding cloned genes are available. Therefore, the development of broadly applicable and reliable transformation markers will be of great importance to fully exploit the enormous potential transgenic insect technology has to offer. Here we review transposon-mediated germ-line transformation approaches that employ green fluorescent protein (GFP) variants to identify successful gene transfer. Furthermore, we provide novel data on the use of DsRed as an additional red fluorescent transformation marker for insect transgenesis. In conclusion, fluorescent proteins controlled by suitable strong promoters possess ideal characteristics to serve as transformation markers for a wide range of insect species.

A transgene-based, embryo-specific lethality system for insect pest management

Biological approaches to insect pest management offer alternatives to pesticidal control. In area-wide control programs that cover entire regions, the sterile insect technique (SIT) can be used to successfully suppress economically important pest species by the mass release of sterilized pest organisms. However, conventional sterilization by ionizing radiation reduces insect fitness, which can result in reduced competitiveness of the sterilized insects. Here we report a transgene-based, dominant embryonic lethality system that allows for generation of large quantities of competitive but sterile insects without the need of irradiation. The system involves the ectopic expression of a hyperactive pro-apoptotic gene that causes embryo-specific lethality when driven by the tetracycline-controlled transactivator (tTA) under the regulation of a cellularization gene enhancer-promoter. We have successfully tested this system in Drosophila melanogaster. The embryonic lethality can be suppressed maternally, which will allow it to be combined with transgenic female-specific lethality systems to raise only vigorous but sterile males.

piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila

P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms.

Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae).

BackgroundThe sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness.ResultsHere, we present a first alternative reproductive sterility system for medfly based on transgenic embryonic lethality. This system is dependent on newly isolated medfly promoter/enhancer elements of cellularization-specifically-expressed genes. These elements act differently in expression strength and their ability to drive lethal effector gene activation. Moreover, position effects strongly influence the efficiency of the system. Out of 60 combinations of driver and effector construct integrations, several lines resulted in larval and pupal lethality with one line showing complete embryonic lethality. This line was highly competitive to wildtype medfly in laboratory and field cage tests.ConclusionThe high competitiveness of the transgenic lines and the achieved 100% embryonic lethality causing reproductive sterility without the need of irradiation can improve the efficacy of operational medfly SIT programs.

Innovations: Applications of insect transgenesis

The recent establishment of broadly applicable genetic transformation systems will allow the analysis of gene function in diverse insect species. This will increase our understanding of developmental and evolutionary biology. Furthermore, insect transgenesis will provide new strategies for insect pest management and methods to impair the transmission of pathogens by human disease vectors. However, these powerful techniques must be applied with great care to avoid harm to our environment.