RESUMO
Mosquitoes constitute the major living beings causing human deaths in the world. They are vectors of malaria, yellow fever, dengue, zika, filariases, chikungunya, among other diseases. New strategies to control/eradicate mosquito populations are based on newly developed genetic manipulation techniques. However, genetic transformation of mosquitoes is a major technical bottleneck due to low efficiency, the need of sophisticated equipment, and highly trained personnel. The present report shows the transgenerational genetic transformation of Aedes aegypti, using the particle inflow gun (PIG), by integrating the ecfp gene in the AAEL000582 mosquito gene with the CRISPR-Cas9 technique, achieving a mean efficiency of 44.5% of bombarded individuals (G0) that showed ECFP expression in their tissues, and a mean of 28.5% transformation efficiency measured on G1 individuals. The same transformation technique was used to integrate the egfp/scorpine genes cloned in the Minos transposon pMinHygeGFP into the Anopheles albimanus genome, achieving a mean efficiency of 43.25% of bombarded individuals (G0) that showed EGFP expression in their tissues. Once the technique was standardized, transformation of Ae. aegypti neonate larvae and An. albimanus eggs was achieved when exposed to gold microparticle bombardment. Integration of genes and heterologous protein expression were confirmed by PCR, sequencing, fluorescent microscopy, mass spectrometry, Western blot and dot blot analyses. Transgenerational inheritance of the transgenes was observed only on Ae. aegypti, as all transformed An. albimanus individuals died at the pupal stage of the G0 generation.
Assuntos
Biolística , Controle de Mosquitos/métodos , Mosquitos Vetores/genética , Transformação Genética , Aedes/genética , Animais , Anopheles/genéticaRESUMO
DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.
Assuntos
Anopheles/genética , Proteínas de Insetos/genética , Animais , Anopheles/crescimento & desenvolvimento , Anopheles/metabolismo , Sequência de Bases , DNA/genética , DNA/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Regiões Promotoras GenéticasRESUMO
Scorpine is an antimicrobial peptide whose structure resembles a hybrid between a defensin and a cecropin. It exhibits antibacterial activity and inhibits the sporogonic development of parasites responsible for murine malaria. In this communication we report the production of scorpine in a heterelogous system, using a specific vector containing its cloned gene. The recombinantly expressed scorpine (RScp) in (Anopheles gambie) cells showed antibacterial activity against (Bacillus subtilis) and (Klebsiella pneumoniae), at 5 and 10 microM, respectively. It also produced 98% mortality in sexual stages of (Plasmodium berghei) at 15 microM and 100% reduction in (Plasmodium falciparum) parasitemia at 5 microM. RScp also inhibited virus dengue-2 replication in C6/36 mosquito cells. In addition, we generated viable and fertile transgenic (Drosophila) that overexpresses and correctly secretes RScp into the insect hemolymph, suggesting that the generation of transgenic mosquitoes resistant to different pathogens may be viable.