Germline Transformation Research Articles

New insight into the mechanism underlying fibroin secretion in silkworm, Bombyxmori.

In order to investigate the role of different parts of the fibroin heavy chain (H-chain) in the secretion of fibroin in the silk gland of the silkworm (Bombyxmori) invivo, two enhanced green fluorescent protein (EGFP)/H-chain fusion genes with deduced protein sequences containing an identical N-terminal region and different C-terminal regions of the H-chain were introduced into the B.mori genome using a piggyBac-mediated germline transformation. EGFP fluorescence and molecular analysis showed the products of two different EGFP/H-chain fusion proteins were secreted into the posterior silk gland lumen and aggregated in the middle silk gland and spun into cocoons. The results revealed that only the non-repetitive N terminus of the H-chain is essential for secretion of the H-chain into the posterior silk gland lumen. In addition, our results also indicated that the most likely post-translational modification of the H-chain is at the C-terminal domain. Here, our results not only provide a theoretical basis for the genetic modification of silk fiber as a functional biomaterial but also are of great significance to establishing a new silk gland bioreactor to mass-produce exogenous proteins in an active form.

Efficient ΦC31 integrase-mediated site-specific germline transformation of Anopheles gambiae.

Current transgenic methodology developed for mosquitoes has not been applied widely to the major malaria vector Anopheles gambiae, which has proved more difficult to genetically manipulate than other mosquito species and dipteran insects. In this protocol, we describe ΦC31-mediated site-specific integration of transgenes into the genome of A. gambiae. The ΦC31 system has many advantages over 'classical' transposon-mediated germline transformation systems, because it allows integration of large transgenes at specific, characterized genomic locations. Starting from a general protocol, we have optimized steps from embryo collection to co-injection of transgene-containing plasmid and in vitro-produced ΦC31 integrase mRNA. We also provide tips for screening transgenic larvae. The outlined procedure provides robust transformation in A. gambiae, resulting in homozygous transgenic lines in ∼2-3 months.

BioSpotlight

BioTechniquesVol. 56, No. 3 BioSpotlightOpen AccessBioSpotlightNathan BlowNathan BlowSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114140AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Bridging the gap in chromatin analysisDetermining the structure and organization of chromatin within cells is important for understanding how gene expression is regulated and how genomes evolve. While a number of techniques, including fluorescence in situ hybridization (FISH) and various chromatin conformation capture analyses (e.g., 3C and Hi-C), have been developed to study chromatin organization and interactions, each of these approaches has drawbacks. While FISH allows single-cell studies of chromatin interactions, the resolution of this technique can be limited to the diffraction limit of light, ∼250 nm. Super resolution microscopy techniques have improved upon this limitation, but still not to sufficient resolution to discern specific chromatin features or enable three-dimensional reconstruction of chromatin interactions. Chromatin conformation capture techniques enable much higher resolution studies of chromatin interactions but also require large numbers of cells, resulting in an inability to assess the frequencies of specific interactions within individual cells. In an effort to bridge the gap between the high resolution of chromatin conformation analysis and the single cell perspective provided by FISH, Chen et al. (Karolinska Institute, Stockholm, Sweden) present a new technique, chromatin in situ proximity (ChrISP), in the current issue of BioTechniques. The methodological basis of ChrISP lies in the well-developed in situ proximity ligation assay (ISPLA), wherein two probes create a fluorescent amplification signal when in close proximity to one another. Two forms of ChrISP were described by the authors: In one, rolling circle amplification (RCA) is used to amplify the signal between the two probes, while the second avoids RCA, instead relying on labeled “splinter” and “backbone” oligos to create a signal between the two chromatin probes. The authors demonstrate that by using the “Green-Splinter ChrISP” (splinter plus backbone oligos with no RCA) they were able to obtain resolutions greater than 17 nm in all 3 dimensions, a 30-fold improvement in the Z-plane in comparison to the other approaches. In the end, given the versatility and simplicity of the ChrISP technique, Chen et al. predict that the method will prove extremely useful when examining chromatin structure alone as well as the alignment of chromatin with other cell landmarks, such as the nuclear membrane and nucleoli.See “Chromatin in situ proximity (ChrISP): Single-cell analysis of chromatin proximities at high resolution”Needle-ing butterfliesAfter years of development and optimization, it is easy for Drosophila researchers to take germline transformation for granted. While it is a demanding technique that quite often requires a large number of injections for success, a transformant can usually be obtained. For other species though, the tools for germline transformation are less well developed, presenting a challenge for scientists wanting to assess genotype/phenotype relationships. But in this issue of BioTechniques, a team of researchers led by Jeffrey Marcus (University of Manitoba, Winnipeg, Canada) report on an improved injection needle design that significantly increases transformation efficiencies and experimental outcomes when working with the buckeye butterfly, Junonia coenia. In pilot injections using a non-optimized needle design previously used for germline transformation in another butterfly species, the authors observed 100% mortality of injected Junonia ova. In an effort to find a needle that would work for Junonia transformation, the group took inspiration from standard Drosophila needles, which have a steep taper and large orifice. Employing these “new” needle designs while injecting Junonia ova resulted in a significantly improved outcome: 21.7% of injected ova hatched with a transformation rate of 3%. Marcus and his team next checked whether or not their new design also showed increased transformation efficiency with other butterfly and moth species beyond Junonia—finding that these needles were indeed more efficient than previous designs. The new needles should be a welcome advance for the large number of researchers working on lepidopteran species where higher transformation efficiency is especially important since fewer ova are available, making genetic analysis a more realistic possibility.See “Improved injection needles facilitate germline transformation of the buckeye butterfly Junonia coenia”A new needle design (Needle type 2) for ova injection during germline transformation experiments of butterfly and moth species significantly improved transformation efficiencies.FiguresReferencesRelatedDetails Vol. 56, No. 3 Follow us on social media for the latest updates Metrics Downloaded 122 times History Published online 3 April 2018 Published in print March 2014 Information© 2014 Author(s)PDF download

Improved Injection Needles Facilitate Germline Transformation of the Buckeye Butterfly Junonia Coenia

Germline transformation with transposon vectors is an important tool for insect genetics, but progress in developing transformation protocols for butterflies has been limited by high post-injection ova mortality. Here we present an improved glass injection needle design for injecting butterfly ova that increases survival in three Nymphalid butterfly species. Using the needles to genetically transform the common buckeye butterfly Junonia coenia, the hatch rate for injected Junonia ova was 21.7%, the transformation rate was 3%, and the overall experimental efficiency was 0.327%, a substantial improvement over previous results in other butterfly species. Improved needle design and a higher efficiency of transformation should permit the deployment of transposon-based genetic tools in a broad range of less fecund lepidopteran species.

Embryonic development of the olive fruit fly, Bactrocera oleae Rossi (Diptera: Tephritidae), in vivo

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is a pest that infests olive fruits. The female oviposits in large green olives and larvae hatch inside the fruit, where they feed upon the fruit tissues. Larval development is completed inside the fruit. These flies cause great damage to olive production worldwide. Traditionally, insecticides have been directed against the adult stage, but the results are not efficient. This present work is a study of embryogenesis in the olive fruit fly. The external morphology of the Bactrocera oleae Rossi (Diptera: Tephritidae) egg is described from light microscopy without dechorionation. The observations were made in vivo and were photographed. The eggshell of B. oleae contains a smooth chorion with a cup-shaped anterior pole. The average length of eggs is 0.738 ± 0.01 mm and the average diameter is 0.21 ± 0.06 mm. The embryonic developmental progress is described as formation of the zygote, blastoderm and gastrulation, and organogenesis. The embryogenesis is completed within 65-70 h at 25 ± 1 °C under laboratory conditions. External egg morphology can be useful in estimating the age of B. oleae eggs for purposes such as introducing genes into embryos by germline transformation in future studies.

Enhanced efficiency of P-element mediated transgenesis in Drosophila: Microinjection of DNA complexed with nanomaterial

The efficiency of genetic transformation technology to generate stable transgenics depends upon the successful delivery of plasmid DNA in embryonic cells. The available gene vectors facilitate efficient plasmid DNA delivery to the cellular milieu but are exposed to nuclease degradation. Recent in vitro studies suggest encapsulation of plasmid DNA with nanomaterial(s) for better protection against nucleases. Therefore, in this study, we tested if complexing of free plasmid DNA with linear polyethylenimine (LPEI, 25 kDa) based nanoparticle (LPN) enhances the efficiency of transformation (transgenesis) by using Drosophila based germ-line transformation technology. Here, we show that the LPN-DNA complex not only enhances the efficiency of this transgenic technology at a DNA concentration of 0.04 μg/μl but also reduces the DNA quantity required to generate transgenics by ten folds. This approach has potential applications for other types of transgenesis and nucleic acid injection methods in Drosophila as well as other popular genetic model systems.

High throughput Agrobacterium tumefaciens-mediated germline transformation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.)

Agrobacterium tumefaciens mediates high frequency of germline transformation of cotton meristem explants. The meristem transformation system we developed is rapid, high throughput and genotype-flexible. We have developed a high throughput cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). The explants were inoculated with a disarmed A. tumefaciens strain, AB33 harboring a 2 T-DNA binary vector pMON114908. This vector contained a gene of interest, an intron-disrupted β-glucuronidase gene in one T-DNA, and a selectable marker gene, aadA in the other T-DNA. Critical factors, such as method of co-culture, culture temperature during selection, composition of selection medium, and selection scheme were found to influence transformation frequency. The cycle time from initial inoculation to the transplanting of transgenic plants to soil was 7-8weeks. Stable integration of transgenes and their transmission to progeny were confirmed by molecular and genetic analyses. Transgenes segregated in the expected Mendelian fashion in the T1 generation for most of the transgenic events. It was possible to recover marker-free events in the T1 generation when utilizing a binary vector that contained the selectable marker and gene of interest expression cassettes on independent T-DNAs. The procedure presented here has been used to regenerate thousands of independent transgenic events from multiple varieties with numerous constructs, and we believe it represents a major step forward in cotton transformation technology.

Conditional targeted genome editing using somatically expressed TALENs in C. elegans

We have developed a method for the generation of conditional knockouts in Caenorhabditis elegans by expressing transcription activator-like effector nucleases (TALENs) in somatic cells. Using germline transformation with plasmids encoding TALENs under the control of an inducible or tissue-specific promoter, we observed effective gene modifications and resulting phenotypes in specific developmental stages and tissues. We further used this method to bypass the embryonic requirement of cor-1, which encodes the homolog of human severe combined immunodeficiency (SCID) protein coronin, and we determined its essential role in cell migration in larval Q-cell lineages. Our results show that TALENs expressed in the somatic cells of model organisms provide a versatile tool for functional genomics.

A novel hAT element in Bombyx mori and Rhodnius prolixus: its relationship with miniature inverted repeat transposable elements (MITEs) and horizontal transfer

Comparative analysis of transposable elements (TEs) from different species can make it possible to reconstruct their history over evolutionary time. In this study, we identified a novel hAT element in Bombyx mori and Rhodnius prolixus with characteristic GGGCGGCA repeats in its subterminal region. Meanwhile, phylogenetic analysis demonstrated that the elements in these two species might represent a separate cluster of the hAT superfamily. Strikingly, a previously identified miniature inverted repeat transposable element (MITE) shared high identity with this autonomous element across the entire length, supporting the hypothesis that MITEs are derived from the internal deletion of DNA transposons. Interestingly, identity of the consensus sequences of this novel hAT element between B. mori and R. prolixus, which diverged about 370 million years ago, was as high as 96.5% over their full length (about 3.6 kb) at the nucleotide level. The patchy distribution amongst species, coupled with overall lack of intense purifying selection acting on this element, suggest that this novel hAT element might have experienced horizontal transfer between the ancestors of B. mori and R. prolixus. Our results highlight that this novel hAT element could be used as a potential tool for germline transformation of R. prolixus to control the transmission of Trypanosoma cruzi, which causes Chagas disease.

Temporal regulation of single-minded target genes in the ventral midline of the Drosophila central nervous system

Differentiation of a specific organ or tissue requires sequential activation of regulatory genes. However, little is known about how serial gene expression is temporally regulated. Here, we present evidence that differential expression of single-minded (sim) target genes can be attributed, in part, to the number of Sim and Tango (Tgo) heterodimer binding sites within their enhancer regions. The Sim, termed a master regulator, directs ventral midline differentiation of Drosophila central nervous system (CNS). According to data on the onset timing of ventral midline gene expression, sim target genes are classified into at least 2 groups (early and late). The sim and rhomboid (rho) genes are activated during early midline differentiation whereas orthodenticle (otd), CG10249, and slit (sli) genes undergo activation during later stages of midline differentiation. Germline transformation and in situ hybridization with transgenic embryos demonstrate that enhancers activating sim and rho expression contain 4 Sim–Tgo binding sites whereas only 1 Sim–Tgo binding site is found in an enhancer of sli. A mutagenized version of the rho enhancer lacking either 1, 2, or 3 Sim–Tgo binding sites mediated progressively more delayed expression of a lacZ reporter gene in the ventral midline. In contrast, a modified sli enhancer displayed progressively earlier onset of lacZ expression when 1, 2, or 3 more Sim–Tgo binding sites were added. Taken together, these results suggest that the number of Sim–Tgo-binding sites is decisive in determining the timing of gene expression in the developing ventral midline. We also discuss a combinatorial model accounting for the sequential expression of sim target genes.

Generation and application of signaling pathway reporter lines in zebrafish

In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.

Germline transformation of the spotted wing drosophilid, Drosophila suzukii, with a piggyBac transposon vector

Drosophila suzukii is a pest of small fruits in many parts of the world, whose management is limited to cultural practices and the use of insecticides. Here we describe a method to genetically manipulate this species in the first step to create female lethality strains useful for the sterile insect technique method of population suppression. This was achieved by the germ-line transformation of D. suzukii with a piggyBac transposon vector having a female-specific lethality effector construct. This can be used in a tetracycline-suppressible conditional gene expression system, when crossed to a suitable tet-transactivator strain. Transformation occurred efficiently, at a frequency of 16% per fertile G0 embryo injected with vector and helper transposase plasmids. The vector was marked for transformant selection with the polyubiquitin-regulated EGFP fluorescent protein, and contains the attP landing site and heterospecific lox recombination sites for post-integration modification of the transgene vector. The 3xP3-AmCyan fluorescent protein marker was inserted within the lox sites to follow a possible recombinase-mediated cassette exchange, that would allow subsequent improvement of the transgenic strain by immobilization of the vector and introduction of new marker cassettes.

Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD600 ≅ 0.6 and diluted to 109 cells ml−1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg ml−1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T0). Among T1 seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ 2) test. Southern hybridization of genomic DNA from kanamycin-resistant T1 transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T1 transgenic plants, thereby confirming stable expression of the nptII gene.

Gene knockout by inducing P-element transposition in Drosophila

The P-element has been successfully used in germline transformation to create transgenic flies and as an insertion mutagenesis agent to disrupt genes. Moreover, P-element can also be used to knockout genes that are near the insertion sites by inducing its imprecise transposition. In this article, P-element insertion lines were crossed with transposase expression fly to recover the transposon, and the deletion of flanking sequence, which was created by imprecise excision, was detected by PCR. Through this method, null alleles of seven genes that spread over three major chromosomes (X, second, third) were generated in Drosophila melanogaster. Results show that the frequency of flanking deletions is expected to be 1%, and it is enough to pick up at least one ideal deletion line from 200 independent recovery lines in general. These data suggest that although the frequency of disrupted gene varied greatly, from 0.13 to 2.34%, gene knockout by inducing P-element transposition appears to be a feasible and effective strategy, compared to the complicated process of gene targeting based on homologous recombination.

Germline Transformation of the Silkworm Bombyx mori L. by Sperm-Mediated Gene Transfer1

The domesticated silkworm Bombyx mori L. has important roles in basic biological research and applied science. To explore the practical use of transgenic technology in agricultural silkworm varieties, we fused the neomycin-resistance gene (Neo(R)) and the green fluorescent protein gene (gfp) into the piggyBac-based transposon vector and transduced it into silkworms by sperm-mediated gene transfer (SMGT). Fluorescence observation indicated the positive rate of G0 egg-batches is 72.7%. After screening against the antibiotic G418, development of individual larvae in the same brood showed significant size differences. PCR detection indicated the existence of gfp and Neo(R) and confirmed the positive rate of transgenesis as 0.47%. Southern blot analysis confirmed the presence of the exogenous genes in the genome of G7 larvae. These results show that our strategy is practical and markedly improves the efficiency of SMGT.

Germline transgenesis of the chordateCiona intestinaliswith hyperactive variants ofsleeping beautytransposable element

Transposon-mediated transgenesis is an excellent method for creating stable transgenic lines and insertional mutants. In the chordate Ciona intestinalis, Minos is the only transposon that has been used as the tool for germline transformation. Adding another transposon system in this organism enables us to conduct genetic techniques which can only be realized with the use of two transposons. In the present study, we found that another Tc1/mariner superfamily transposon, sleeping beauty (SB), retains sufficient activity for germline transformation of C. intestinalis. SB shows efficiencies of germline transformation, insertion into gene coding regions, and enhancer detection comparable to those of Minos. We have developed a system for the remobilization of SB copies in the C. intestinalis genome by using transgenic lines expressing SB transposase in the germ cells. With this system, we examined the manner of SB mobilization in the C. intestinalis genome. SB shows intrachromosomal transposition more frequently than Minos. SB-based germline transformation and the establishment of a new method that uses its frequent intrachromosomal transposition will result in breakthroughs in genetic approaches that use C. intestinalis together with Minos.

Ovine PrP transgenic Drosophila show reduced locomotor activity and decreased survival

Drosophila have emerged as a model system to study mammalian neurodegenerative diseases. In the present study we have generated Drosophila transgenic for ovine PrP (prion protein) to begin to establish an invertebrate model of ovine prion disease. We generated Drosophila transgenic for polymorphic variants of ovine PrP by PhiC31 site-specific germ-line transformation under expression control by the bi-partite GAL4/UAS (upstream activating sequence) system. Site-specific transgene insertion in the fly genome allowed us to test the hypothesis that single amino acid codon changes in ovine PrP modulate prion protein levels and the phenotype of the fly when expressed in the Drosophila nervous system. The Arg(154) ovine PrP variants showed higher levels of PrP expression in neuronal cell bodies and insoluble PrP conformer than did His(154) variants. High levels of ovine PrP expression in Drosophila were associated with phenotypic effects, including reduced locomotor activity and decreased survival. Significantly, the present study highlights a critical role for helix-1 in the formation of distinct conformers of ovine PrP, since expression of His(154) variants were associated with decreased survival in the absence of high levels of PrP accumulation. Collectively, the present study shows that variants of the ovine PrP are associated with different spontaneous detrimental effects in ovine PrP transgenic Drosophila.

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori.

Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

Germline transformation of the diamondback moth, Plutella xylostella L., using the piggyBac transposable element

The diamondback moth, Plutella xylostella, is one of the most economically important agricultural pests. The larvae of this moth cause damage by feeding on the foliage of cruciferous vegetables such as cabbage, broccoli, cauliflower and rapeseed. Control generally comprises chemical treatment; however, the diamondback moth is renowned for rapid development of resistance to pesticides. Other methods, such as biological control, have not been able to provide adequate protection. Germline transformation of pest insects has become available in recent years as an enabling technology for new genetics-based control methods, such as the Release of Insects carrying a Dominant Lethal (RIDL(®) ). In the present study, we report the first transformation of the diamondback moth, using the piggyBac transposable element, by embryo microinjection. In generating transgenic strains using four different constructs, the function of three regulatory sequences in this moth was demonstrated in driving expression of fluorescent proteins. The transformation rates achieved, 0.48-0.68%, are relatively low compared with those described in other Lepidoptera, but not prohibitive, and are likely to increase with experience. We anticipate that germline transformation of the diamondback moth will permit the development of RIDL strains for use against this pest and facilitate the wider use of this species as a model organism for basic studies.

Infertility resulting from transgenic I-PpoI male Anopheles gambiae in large cage trials

Objectives: Anopheles gambiae is the primary vector of malaria in sub-Saharan Africa and is a potential target of genetic control programs. We determined the capacity of male A. gambiae created by germline transformation to introduce infertility into stable age-distribution populations. We also determined effects of the transgenes on life history.Methods: Stable age-distribution populations of A. gambiae mosquitoes were established in large indoor cages. Male mosquitoes carrying an I-PpoI homing endonuclease gene were introduced at ×5 and ×10 release rates where they competed with target male mosquitoes for matings. Similar trials were conducted in small cages with an additional ×1 release level.Results: Infertility was successfully introduced into all target populations. In supporting experiments, complete female infertility was observed in all strains and species of the A. gambiae complex to which transgenic males were mated. Life history experiments demonstrated that reductions in I-PpoI male vigor exist in the form of reduced adult male emergence, longevity and competitiveness.Discussion: A. gambiae I-PpoI males are capable of introducing high levels of infertility in target populations in indoor cage trials. This was accomplished despite losses of vigor resulting from the HEG transgene. These results motivate further trials of sexually I-PpoI A. gambiae in outdoor cage and field trials.