Reduction of malaria transmission by transgenic mosquitoes expressing an antisporozoite antibody in their salivary glands

We have previously developed a robust salivary gland‐specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti‐P. falciparum circumsporozoite protein (PfCSP) single‐chain antibody (scFv) fused to DsRed in a secretory form (mDsRed‐2A10 scFv). Fluorescence microscopy showed that the mDsRed‐2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission‐blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed‐2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland‐specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans.     

Transgenic characterization of two testis‐specific promoters in the silkworm,Bombyx mori

Sex‐specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female‐specific modification system whereas little success was reported on male‐specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene‐based, female‐specific lethality system has been established based on sex‐specific alternative splicing factors and a female‐specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male‐specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis‐specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta‐tubulin 4 gene (Bmβ4) were introduced using piggybac‐based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis‐specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis‐specific gene expression. Identification of these testis‐specific promoters not only contributes to a better understanding of testis‐specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.     

Honey bee promoter sequences for targeted gene expression

The honey bee, Apis mellifera, displays a rich behavioural repertoire, social organization and caste differentiation, and has an interesting mode of sex determination, but we still know little about its underlying genetic programs. We lack stable transgenic tools in honey bees that would allow genetic control of gene activity in stable transgenic lines. As an initial step towards a transgenic method, we identified promoter sequences in the honey bee that can drive constitutive, tissue‐specific and cold shock‐induced gene expression. We identified the promoter sequences of Am‐actin5c, elp2l, Am‐hsp83 and Am‐hsp70 and showed that, except for the elp2l sequence, the identified sequences were able to drive reporter gene expression in Sf21 cells. We further demonstrated through electroporation experiments that the putative neuron‐specific elp2l promoter sequence can direct gene expression in the honey bee brain. The identification of these promoter sequences is an important initial step in studying the function of genes with transgenic experiments in the honey bee, an organism with a rich set of interesting phenotypes.     

Two-dimensional gel analysis of haemolymph proteins from Plasmodium-melanizing and -non-melanizing strains of Anopheles gambiae

Haemolymph polypeptides from Plasmodium‐refractory and ‐susceptible mosquitoes were compared by one‐ and two‐dimensional gel electrophoresis. The refractory strain of Anopheles gambiae kills malaria parasites by a humoral melanization mechanism whereas the parasites develop normally in susceptible mosquitoes. The two strains respond in a similar manner to carboxy‐methyl‐Sephadex beads that have been injected into the thoracic haemocoel, i.e. beads are strongly melanized in refractory but not susceptible mosquitoes. Protein profiles were compared between strains following cold shock (naïve control), saline injection and Sephadex bead injection. Using the susceptible naïve control as the standard, eight constitutively expressed polypeptides were specific to naïve susceptible mosquitoes while twelve other spots were reduced, enhanced or specific to refractory mosquitoes. Several of the strain‐specific spots probably comprise related pairs (one in each strain) which vary only in isoelectric focusing point. Nine spots were induced by sham injection or by an injection of beads or saline, but none was reproducibly different between the strains. Amino acid sequence analysis of one of the refractory strain‐specific spots identified it as AgSp14D1, an A. gambiae infection‐responsive serine protease that is most similar to the Drosophila gene easter and Manduca prophenoloxidase activating enzyme. This gene maps to polytene chromosome division 14, which has been implicated in the melanization phenotype by quantitative trait loci mapping.     

Molecular basis for odorant receptor tuning: a short C‐terminal sequence is necessary and sufficient for selectivity of mosquito Or8

A birth‐and‐death evolutionary model for odorant receptor gene repertoires presumes the creation of repertoires with the capacity for high‐level diversity and rapid ligand specificity change. This changes the recognised odour space, directly affecting fitness‐related behaviours and ultimately affecting adaptation to new environments and resources. The proximate molecular mechanisms underlying the tuning of odorant receptor repertoires, and thus peripheral olfaction, are unclear. In the present study, we report a concrete example of this model of odorant receptor evolution leading to rapid changes in receptor tuning that leave the peripheral neuronal circuitry intact. We identified a conserved odorant receptor gene in mosquitoes, Or8, which in Culex quinquefasciatus underwent a duplication and inversion event. The paralogues differ in only minor structural changes manifesting at the C‐terminus. We assessed the specificity of the paralogous odorant receptors and receptor neurones. We found that the functional tuning of the receptor was indeed reflected in minor differences in amino acid structure. Specifically, we found that enantiomeric specificity of these mosquito Or8 paralogues relies on eight C‐terminal amino acids encoded in the final exon of the gene; thus, the birth of a paralogous odorant receptor can change the tuning of the peripheral olfactory system.     

Modular cis‐regulatory logic of yellow gene expression in silkmoth larvae

Colour patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (ie yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remain poorly understood. To elucidate this, molecular studies involving dipterans have largely analysed the cis‐regulatory regions of pigmentation genes and have revealed cis‐regulatory modularity. Here, we used well‐developed transgenic techniques in Bombyx mori and demonstrated that cis‐regulatory modularity controls tissue‐specific expression of the yellow gene. We first identified which body parts are regulated by the yellow gene via black pigmentation. We then isolated three discrete regulatory elements driving tissue‐specific gene expression in three regions of B. mori larvae. Finally, we found that there is no apparent sequence conservation of cis‐regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans‐regulatory landscapes of the yellow gene differ significantly between the two taxa. The results of this study confirm that lepidopteran species use cis‐regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms.     

Evolution of the neural sex‐determination system in insects: does fruitless homologue regulate neural sexual dimorphism in basal insects?

In the brain of holometabolous insects such as the fruit fly Drosophila melanogaster, the fruitless gene produces sex‐specific gene products under the control of the sex‐specific splicing cascade and contributes to the formation of the sexually dimorphic circuits. Similar sex‐specific gene products of fruitless homologues have been identified in other holometabolous insects such as mosquitoes and a parasitic wasp, suggesting the fruitless‐dependent neural sex‐determination system is widely conserved amongst holometabolous insects. However, it remains obscure whether the fruitless‐dependent neural sex‐determination system is present in basal hemimetabolous insects. To address this issue, identification, characterization, and expression analyses of the fruitless homologue were conducted in the two‐spotted cricket, Gryllus bimaculatus, as a model hemimetabolous insect. The Gryllus fruitless gene encodes multiple isoforms with a unique zinc finger domain, and does not encode a sex‐specific gene product. The Gryllus Fruitless protein is broadly expressed in the neurones and glial cells in the brain, and there was no prominent sex‐related difference in the expression levels of Gryllus fruitless isoforms. The results suggest that the Gryllus fruitless gene is not involved in the neural sex‐determination in the cricket brain.     

Functional characterization of the vitellogenin promoter in the silkworm,Bombyx mori

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis‐regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798‐bp DNA sequence adjacent to the 5′‐end of the vitellogenin gene (Bmvg). PiggyBac‐based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex‐, tissue‐ and stage‐specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval?pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20‐hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis‐regulatory element in B. mori.     

Functional factor VIII made with von Willebrand factor at high levels in transgenic milk

Summary. Background: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives: To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods: Transgenic mice were made with a mammary‐specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS‐PAGE, western blot, and one‐stage and two‐stage clotting assays. The hemostatic activity of immunoaffinity‐enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions: With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single‐chain molecule that retained high specific activity, similar to therapeutic‐grade FVIII. 226/N6 had > 450‐fold higher IU mL^?1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma‐derived VWF as therapeutic‐grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof‐of‐principle for the study of expression of 226/N6 and perhaps other single‐chain bioengineered rFVIIIs in the milk of transgenic livestock.     

Transgene‐mediated suppression of the RNA interference pathway in Aedes aegypti interferes with gene silencing and enhances Sindbis virus and dengue virus type 2 replication

RNA interference (RNAi) is the major innate antiviral pathway in Aedes aegypti that responds to replicating arboviruses such as dengue virus (DENV) and Sindbis virus (SINV). On the one hand, the mosquito's RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. On the other, transient silencing of key genes of the RNAi pathway increases replication of SINV and DENV2, allowing the viruses to temporally overcome dose‐dependent midgut infection and midgut escape barriers (MEB) more efficiently. Here we expressed Flock house virus B2 (FHV‐B2) from the poly‐ubiquitin (PUb) promoter in Ae. aegypti using the ΦC31 site‐directed recombination system to investigate the impact of transgene‐mediated RNAi pathway suppression on infections with SINV‐TR339eGFP and DENV2‐QR94, the latter of which has been shown to be confronted with a strong MEB in Ae. aegypti. FHV‐B2 was constitutively expressed in midguts of sugar‐ and blood‐fed mosquitoes of transgenic line PUbB2 P61. B2 over‐expression suppressed RNA silencing of carboxypeptidase A‐1 (AeCPA‐1) in midgut tissue of PUbB2 P61 mosquitoes. Following oral challenge with SINV‐TR339eGFP or DENV2‐QR94, mean titres in midguts of PUbB2 P61 females were significantly higher at 7 days post‐bloodmeal (pbm) than in those of nontransgenic control mosquitoes. At 14 days pbm, infection rates of carcasses were significantly increased in PubB2 P61 mosquitoes infected with SINV‐TR339eGFP. Following infection with DENV2‐QR94, midgut infection rates were significantly increased in the B2‐expressing mosquitoes at 14 days pbm. However, B2 expression in PUbB2 P61 did not increase the DENV2‐QR94 dissemination rate, indicating that the infection phenotype was not primarily controlled by RNAi.     

Identification and characterization of a putative sevenless homologue in the malaria vector Anopheles gambiae

Tyrosine kinase sequences were identified and characterized in Anopheles gambiae, the major vector of malaria in subsaharan Africa. One of these sequences has the characteristics expected for a homologue of the Drosophila sevenless gene, which is necessary for R7 photoreceptor cell fate determination in the developing compound eye. The putative Anopheles seven-less gene homologue is located in a telomeric region of the X chromosome and is expressed in the head of late larval and pupal stage mosquitoes. Identification of the Anopheles homologue of the sevenless gene is a first step towards the development of a dominant phenotypic marker that could be used for detecting transformed Anopheles mosquitoes in a wide variety of genetic backgrounds and, as such, could be used in the development of transgenic mosquitoes for the control of parasite transmission. Preliminary evidence for sevenless sequences were also found in DNA from blackfly, Mediterranean fruit fly and the honeybee.     

Flying vaccinator; a transgenic mosquito delivers a Leishmania vaccine via blood feeding

‘Flying vaccinator’ is the concept of using genetically engineered hematophagous insects to deliver vaccines. Here we show the generation of a transgenic anopheline mosquito that expresses the Leishmania vaccine candidate, SP15, fused to monomeric red fluorescent protein (mDsRed) in its salivary glands. Importantly, mice bitten repeatedly by the transgenic mosquitoes raised anti‐SP15 antibodies, indicating delivery of SP15 via blood feeding with its immunogenicity intact. Thus, this technology makes possible the generation of transgenic mosquitoes that match the original concept of a ‘flying vaccinator’. However, medical safety issues and concerns about informed consent mitigate the use of the ‘flying vaccinator’ as a method to deliver vaccines. We propose that this expression system could be applied to elucidate saliva–malaria sporozoite interactions.     

Knockdown of mitogen‐activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos

Arthropod‐borne infectious diseases are responsible for nearly 1.5 million deaths annually across the globe, with malaria responsible for >50% of these deaths. Recent efforts to enhance malaria control have focused on developing genetically modified Anopheles mosquitoes that are resistant to malaria parasite infection by manipulating proteins that are essential to the immune response. Although this approach has shown promise, the lack of efficient genetic tools in the mosquito makes it difficult to investigate innate immunity using reverse genetics. Current gene knockdown strategies based on small interfering RNA are typically labourious, inefficient, and require extensive training. In the present study, we describe the use of morpholino antisense oligomers to knockdown MEK‐ERK signalling in the midgut of Anopheles stephensi through a simple feeding protocol. Anti‐MEK morpholino provided in a saline meal was readily ingested by female mosquitoes with minimal toxicity and resulted in knockdown of total MEK protein levels 3–4 days after morpholino feeding. Further, anti‐MEK morpholino feeding attenuated inducible phosphorylation of the downstream kinase ERK and, as predicted by previous work, reduced parasite burden in mosquitoes infected with Plasmodium falciparum. To our knowledge, this is the first example of morpholino use for target protein knockdown via feeding in an insect vector. Our results suggest this method is not only efficient for studies of individual proteins, but also for studies of phenotypic control by complex cell signalling networks. As such, our protocol is an effective alternative to current methods for gene knockdown in arthropods.     

Local chromatin interactions contribute to expression of the fibrinogen gene cluster

Essentials

• The fibrinogen gene cluster is flanked by CCCTC‐binding factor (CTCF) interaction sites.
• Chromatin looping of the fibrinogen cluster was demonstrated by chromosome conformation capture.
• Deleting a CTCF interaction site alters chromatin looping and halves fibrinogen expression.
• Looping of the human fibrinogen locus is functionally linked to fibrinogen gene expression.

Summary

Background

The coordinately regulated genes encoding human fibrinogen are clustered. This evolutionarily conserved configuration provides a possible mechanism for co‐regulation whereby regulatory elements influence gene expression locally. The cluster is flanked by CCCTC‐binding factor (CTCF) interaction sites that are candidate insulator regions mediating chromatin looping.

Objectives

To further our understanding of fibrinogen gene regulation, we aimed to investigate whether interactions exist between parts of the fibrinogen locus and how these contacts contribute to fibrinogen expression.

Methods

We used chromosome conformation capture in cultured cell lines to detect chromatin interactions at the fibrinogen gene cluster. We generated clonal cell lines where two CTCF interaction sites at one end of the locus were deleted using CRISPR‐Cas9‐mediated genome editing. Fibrinogen expression and protein production were measured using qRT‐PCR and ELISA, respectively.

Results

We detected proximity between the ends of the fibrinogen locus, regardless of whether cells express fibrinogen. An interaction between the FGA promoter and the edge of the locus was more frequent in fibrinogen‐expressing cells. Deletion of a CTCF site at one edge of the cluster altered chromatin interactions, reduced steady‐state expression of FGB and FGG mRNA, and led to a halving of secreted fibrinogen. These phenotypes were completely restored by reintroduction of the CTCF interaction motif in previously motif‐deleted clones.

Conclusions

Chromatin interactions are important for the coordinated regulation of the human fibrinogen genes. This finding furthers our comprehension of how fibrinogen is produced and identifies a possible source of variability in plasma fibrinogen levels seen in populations.