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.     

Robust salivary gland-specific transgene expression in Anopheles stephensi mosquito

Malaria sporozoites invade the mosquito salivary glands and wait in the salivary duct until the next blood feeding. The mechanisms of the process and molecules involved in the salivary gland invasion remain largely unknown. To establish a robust salivary gland-specific transgene expression in Anopheles stephensi, we obtained a salivary gland-specific promoter for a gene encoding anopheline antiplatelet protein (AAPP). The aapp promoter is a female salivary gland-specific and blood meal-inducible strong promoter. Using this promoter, we generated a transgenic An. stephensi expressing abundant Discosoma sp. red fluorescent protein (DsRed) in the distal-lateral lobes of the glands, where the sporozoites invade preferentially. These results open up the possibilities of elucidating salivary gland-parasite interactions and generating transgenic mosquitoes refractory to parasites.     

Axolotl: A resourceful vertebrate model for regeneration and beyond

The regenerative capacity varies significantly among the animal kingdom. Successful regeneration program in some animals results in the functional restoration of tissues and lost structures. Among the highly regenerative animals, axolotl provides multiple experimental advantages with its many extraordinary characteristics. It has been positioned as a regeneration model organism due to its exceptional renewal capacity, including the internal organs, central nervous system, and appendages, in a scar-free manner. In addition to this unique regeneration ability, the observed low cancer incidence, its resistance to carcinogens, and the reversing effect of its cell extract on neoplasms strongly suggest its usability in cancer research. Axolotl's longevity and efficient utilization of several anti-aging mechanisms underline its potential to be employed in aging studies.     

In vivo expression of a conditional TGF-beta1 transgene: no evidence for TGF-beta1 transgene expression in SM22alpha-tTA transgenic mice

Transforming growth beta-1 (TGF-beta1) appears to play a critical role in the regulation of arterial intimal growth and the development of atherosclerosis. TGF-beta1 is expressed at increased levels in diseased arteries; however, its role in disease development remains controversial. Experiments in which TGF-beta1 is overexpressed in the artery wall of transgenic mice could clarify the role of TGF-beta1 in the development or prevention of vascular disease. However, constitutive overexpression of a TGF-beta1 transgene in the mouse artery wall is embryonically lethal. Therefore, to overexpress TGF-beta1 in the artery wall of adult mice, we generated mice that were transgenic for a conditional, tetracycline operator (tetO)-driven TGF-beta1 allele. These mice were viable, and when crossed with mice expressing a tetracycline-regulated transactivator (tTA) in the heart, expressed the TGF-beta1 transgene in a cardiac-restricted and doxycycline-dependent manner. Nevertheless, breeding of the tetO-TGF-beta1 transgene into three lines of mice transgenic for a smooth muscle-targeted tTA (SM22alpha-tTA mice; reported elsewhere to transactivate tetO-driven alleles in smooth muscle cells of large arteries) did not yield expression of the TGF-beta1 transgene. Moreover, tTA expression was not detected in aortae of the SM22alpha-tTA mice. Transgenic mice that express tTA at high levels in vascular smooth muscle and reliably transactivate tetO-driven transgenes would be useful for deciphering the role of TGF-beta1 (or other proteins) in normal arterial physiology and in the development of arterial disease. Currently available SM22alpha-tTA mice were not useful for this purpose. Generation of higher-expressing lines of SM22alpha-tTA mice appears warranted.     

Induction of antisporozoite antibodies by biting of transgenic Anopheles stephensi delivering malarial antigen via blood feeding

We produced a transgenic mosquito expressing a rodent malaria vaccine candidate antigen in the salivary gland. Three tandemly repeated amino acid units from the repeat region of circumsporozoite protein of Plasmodium berghei (PbCS3R) fused to red fluorescent protein (monomeric DsRed) was chosen as a vaccine candidate antigen. Immunoblot and fluorescence microscopic analyses showed the transgene expression in the female salivary gland. The transgene product was released from the proboscis as a component of saliva. The monomeric DsRed-fusion expression system could be suitable for transgene secretion in the saliva of female mosquitoes. Mice repeatedly bitten by transgenic mosquitoes raised antibodies against P. berghei sporozoites, and the sera had protective ability against sporozoite invasion of human hepatoma HepG2 cells. These results suggest that transgene products are immunogenically active in saliva, and induce the antibodies to malaria parasite. These findings indicate that this technology has the potential for production of a 'flying vaccinator' for rodent malaria parasites.     

Transgenic mice for conditional gene manipulation in astroglial cells

Astrocytes are thought to exert diverse functions in the brain, but it has been difficult to prove this in vivo because of a scarcity of tools to manipulate these cells. Here, we report the generation of new transgenic mouse lines that allow for conditional gene ablation in astrocytes using the tamoxifen- (TAM-) inducible CreER(T2)/loxP system and bacterial artificial chromosome (BAC)-based transgenesis. In adult transgenic mice, where CreER(T2) expression is driven by the promoter of the sodium-dependent glutamate/aspartate transporter (Glast/Slc1a3) or of connexin 30 (Cx30/Gjb6), intraperitoneal TAM-injection induced Cre-mediated recombination in astroglial cells throughout the brain. Targeting efficacies varied in a region-specific manner from 20 to 90% as indicated by enzyme-based reporter lines and immunohistochemical staining. In addition, the Glast-line allowed to target retinal Müller cells and adult neural stem/progenitor cells in neurogenic regions of the adult brain. Transgenic mice expressing CreER(T2) under the control of the apolipoprotein e (ApoE) or aquaporin 4 (Aqp4) promoter showed inducible recombination in different areas of the central nervous system (CNS) albeit at low levels. Transgenic lines showed TAM-induced recombination in specific peripheral organs. These new mouse lines should help to further explore the relevance of astrocytes for brain function, as well as their contribution to pathological conditions because of aging, disease or injury.     

Ex Vivo Programming of Antigen-Presenting B Lymphocytes: Considerations on DNA Uptake and Cell Activation

Plasmids used in DNA vaccination not only serve as a source of antigen, but also have an important adjuvant effect. This review focuses on recent advancements made in understanding how cells internalize DNA, and how internalized DNA activates immune response pathways. We also comment on the role of B cells in both of these processes.     

Collagen-binding protein,Aegyptin, regulates probing time and blood feeding success in the dengue vector mosquito,Aedes aegypti

Mosquito salivary glands have important roles in blood feeding and pathogen transmission. However, the biological relevance of many salivary components has yet to be determined. Aegyptin, a secreted salivary protein from Aedes aegypti, binds collagen and inhibits platelet aggregation and adhesion. We used a transgenic approach to study the relevance of Aegyptin in mosquito blood feeding. Aedes aegypti manipulated genetically to express gene-specific inverted-repeat RNA sequences exhibited significant reductions in Aegyptin mRNA accumulation (85–87%) and protein levels (>80-fold) in female mosquito salivary glands. Transgenic mosquitoes had longer probing times (78–300 s, P < 0.0001) when feeding on mice compared with controls (15–56 s), feeding success was reduced, and those feeding took smaller blood meals. However, no differences in feeding success or blood meal size were found in membrane feeding experiments using defibrinated human blood. Salivary gland extracts from transgenic mosquitoes failed to inhibit collagen-induced platelet aggregation in vitro. Reductions of Aegyptin did not affect salivary ADP-induced platelet aggregation inhibition or disturb anticlotting activities. Our results demonstrate the relevance of Aegyptin for A. aegypti blood feeding, providing further support for the hypothesis that platelet aggregation inhibition is a vital salivary function in blood feeding arthropods. It has been suggested that the multiple mosquito salivary components mediating platelet aggregation (i.e., Aegyptin, apyrase, D7) represent functional redundancy. Our findings do not support this hypothesis; instead, they indicate that multiple salivary components work synergistically and are necessary to achieve maximum blood feeding efficiency.Aedes aegypti, the main vector of dengue, chikungunya, and yellow fever viruses, is an anautogenous mosquito that requires a blood meal for egg development. Female mosquitoes probe the skin of a suitable vertebrate host to acquire a blood meal. Hematophagy (blood feeding) to repletion usually takes from seconds to a few minutes (1). The stylets used to pierce the vascular beds of the vertebrate hosts cause local damage that triggers host hemostatic responses.Primary hemostasis is characterized by vascular contraction, platelet adhesion, and formation of a soft aggregate plug that begins immediately after endothelial disruption. Injury causes temporary local contraction of vascular smooth muscle. Vasoconstriction slows blood flow, enhancing platelet adhesion and activation. The soft platelet plug is stabilized to form a clot during secondary hemostasis (2, 3). Hematophagic arthropods have evolved a complex mixture of salivary components that counteract platelet aggregation, blood coagulation, vasoconstriction, and inflammation to overcome this efficient and redundant vertebrate hemostatic system (4–6).Aedes aegypti, Anopheles stephensi, and Simulim nigrimanum express salivary collagen-binding proteins that prevent collagen-induced platelet aggregation and adhesion by blocking its interaction with the platelet receptors glycoprotein VI and Integrin α₂β₁, and von Willebrand factor (vWF) (7–10). Aegyptin is an A. aegypti salivary protein that binds directly through its C-terminal domain to the vWF binding site in collagen (7, 8). Moreover, Aegyptin prevents carotid thrombus formation in vivo without causing excessive bleeding in mice.We used a transgenic approach to reduce the products of the Aegyptin gene (also known as the 30K b gene; ref. 11) to study its relevance in blood feeding. Our results show that reduced levels of Aegyptin mRNAs and protein result in a phenotype in which the probing times of transgenic mosquitoes are increased significantly, and their salivary gland extracts fail to inhibit collagen-induced platelet aggregation in vitro. Reductions in Aegyptin did not affect either the salivary gland anticoagulant or ADP-induced platelet aggregation activities. We interpret these data to indicate that the altered feeding capabilities of transgenic insects results from their inability to inhibit collagen-induced platelet aggregation.Although mosquito salivary antihemostatic activity has been demonstrated, it remains to be proven whether each particular salivary component is relevant or necessary to acquire a successful blood meal. It was proposed that redundancy of salivary function reinforces the efficiency of blood feeders (12, 13). However, our results do not support this general hypothesis. It appears that the diversity and complexity of antihemostatic proteins found in salivary secretions is not entirely redundant because reduction of Aegyptin alone impairs significantly the ability of the mosquito to acquire a blood meal.