Comparison of transgene expression in Aedes aegypti generated by mariner Mos1 transposition and ΦC31 site-directed recombination.

Transgenic mosquitoes generated by transposable elements (TEs) often poorly express transgenes owing to position effects. To avoid these effects, the ΦC31 site-directed recombination system was used to insert transgenes into a locus favourable for gene expression in Aedes aegypti. We describe phenotypes of mariner Mos1 TE and ΦC31 transgenic mosquitoes expressing the enhanced green fluorescent protein (EGFP) reporter in midguts of blood-fed females. Mosquitoes of nine TE-generated lines [estimated transformation frequency (TF): 9.3%] clearly expressed the eye-specific selection marker but only 2/9 lines robustly expressed the EGFP reporter. The piggyBac TE-generated ΦC31 docking strain, attP26, supported recombination with attB site containing donors at an estimated TF of 1.7-4.9%. Using a codon-optimized ΦC31 integrase mutant instead of the 'wild-type' enzyme did not affect TF. Site-directed recombination of line attP26 with an attB-containing donor expressing EGFP from the Ae. aegypti carboxypeptidase promoter produced one transgenic line with blood-fed females expressing the reporter in midgut tissue. Docking strain attP26 also supported robust expression of Flock House virus B2 from the Ae. aegypti polyubiquitin promoter. Our data confirm that eye-specific selection marker expression alone is not a reliable indicator for robust gene-of-interest expression in Ae. aegypti and that the ΦC31 system can ensure predictable transgene expression in this mosquito species.

Transgene-mediated suppression of dengue viruses in the salivary glands of the yellow fever mosquito, Aedes aegypti.

Controlled sex-, stage- and tissue-specific expression of antipathogen effector molecules is important for genetic engineering strategies to control mosquito-borne diseases. Adult female salivary glands are involved in pathogen transmission to human hosts and are target sites for expression of antipathogen effector molecules. The Aedes aegypti 30K a and 30K b genes are expressed exclusively in adult female salivary glands and are transcribed divergently from start sites separated by 263 nucleotides. The intergenic, 5'- and 3'-end untranslated regions of both genes are sufficient to express simultaneously two different transgene products in the distal-lateral lobes of the female salivary glands. An antidengue effector gene, membranes no protein (Mnp), driven by the 30K b promoter, expresses an inverted-repeat RNA with sequences derived from the premembrane protein-encoding region of the dengue virus serotype 2 genome and reduces significantly the prevalence and mean intensities of viral infection in mosquito salivary glands and saliva.

Stability and loss of a virus resistance phenotype over time in transgenic mosquitoes harbouring an antiviral effector gene.

Transgenic Aedes aegypti were engineered to express a virus-derived, inverted repeat (IR) RNA in the mosquito midgut to trigger RNA interference (RNAi) and generate resistance to dengue virus type 2 (DENV2) in the vector. Here we characterize genotypic and phenotypic stabilities of one line, Carb77, between generations G(9) and G(17). The anti-DENV2 transgene was integrated at a single site within a noncoding region of the mosquito genome. The virus resistance phenotype was strong until G(13) and suppressed replication of different DENV2 genotypes. From G(14)-G(17) the resistance phenotype to DENV2 became weaker and eventually was lost. Although the sequence of the transgene was not mutated, expression of the IR effector RNA was not detected and the Carb77 G(17) mosquitoes lost their ability to silence the DENV2 genome.

Development of a new Sindbis virus transducing system and its characterization in three Culicine mosquitoes and two Lepidopteran species.

Alphavirus transducing systems (ATSs) are alphavirus-based tools for expressing genes in insects. Here we describe an ATS (5'dsMRE16ic) based entirely on Sindbis MRE16 virus. GFP expression was used to characterize alimentary tract infections and dissemination in three Culicine and two Lepidopteran species. Following per os infection, 5'dsMRE16ic-EGFP efficiently infected Aedes aegypti and Culex tritaeniorhynchus, but not Culex pipiens pipiens. Ae. aegypti clearly showed accumulation of green fluorescent protein (GFP) in the posterior midgut and foregut/midgut junction within 2-3 days postinfection. Following parenteral infection of larvae, Bombyx mori had extensive GFP expression in larvae and adults, but Manduca sexta larvae were mostly resistant. 5'dsMRE16ic should be a valuable tool for gene expression in several important insect species that are otherwise difficult to manipulate genetically.

The P450 aromatase (P450 arom) gene is asymmetrically expressed in a critical period for gonadal sexual differentiation in the chick.

Steroid hormones appear to play an important role in gonadal sex differentiation of birds. Here we studied the steady-state level of the P450 arom mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) in the left and the right presumptive ovary and testis of developing chicken embryos. The gonads were evaluated every hour during the undifferentiated period, at 144-156 h of incubation (h/i), and every 24 h after sexual differentiation at 168 and 192 h/i. Activity of P450 arom was determined by estrone production from [3H]androstenedione at 144-192 h/i. Moreover, morphological development of the gonad was also examined by light microscopy. Results show that onset of P450 arom mRNA and its protein activity were simultaneously detected in the left and the right ovaries at 147 h/i. Asymmetric function of P450 arom gene expression was observed at 156 h/i when morphological gonadal differentiation is first recognized. Biotransformation of [3H]androstenedione to estrone was also asymmetrically detected between the left and right gonad at 156 h/i and asymmetry was maintained throughout the analyzed stages. It is proposed that there is a gene in birds that is asymmetrically expressed in the undifferentiated stage of the female and the male gonad. In the female this gene could promote P450 arom gene expression, increasing estrogen production, which in turn could induce ovarian cortex proliferation and expression of other structural estrogen-regulated genes involved in ovarian sexual determination.