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First published online September 23, 2003

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Transgenesis and reverse genetics of mosquito innate immunity

Sang Woon Shin, Vladimir A. Kokoza and Alexander S. Raikhel^*

Department of Entomology, University of California, Riverside, California 92521, USA

View larger version (14K): [in a new window]   Fig. 1. Schematic illustration of the regulatory regions of Ae. aegypi Vitellogenin (Vg) gene. Numbers refer to nucleotide positions relative to the transcription start site, and letters refer to restriction enzyme sites: E, EcoRI; R, EcoRV; K, KpnI; S, Sau3A. C/EBP, response element of C/EBP transcription factor; EcRE, ecdysteroid response element; E74 and E75, response elements for the respective early gene product of the ecdysone hierarchy; GATA, response element for GATA transcription factor; HNF-3/fkh, response element for HNF/forkhead factor; Vg, coding region of the Vg gene. (Reproduced from Kokoza et al., 2001a, with permission.)

View larger version (46K): [in a new window]   Fig. 2. Transgenic mosquitoes with the 3xP3-EGFP selectable marker and structure of the transformation vector overexpressing the immune effector molecules and its expression in transgenic mosquitoes. (A) Expression of the 3xP3-EGFP selectable marker was observed in the eyes of the larval, pupal and adult stages of transgenic Ae. aegypti. (B) Schematic diagram of the pBac[3xP3-EGFP afm, DefA or CecA] transformation vector that was transformed into the Ae. aegypti germ line. (C) Developmental profiles of Vg-DefA and Vg mRNA expression in the fat bodies of the transgenic mosquitoes. The DefA peptide level of hemolymph was detected by western analysis. PV, previtellogenic stage. (D) The increased resistance to Enterobacter cloacae was shown by the survival test of transgenic mosquitoes. Survival rates (%) of the parental wild-type and transgenic blood-fed mosquitoes at 24 h after the injection of E. cloacae are shown. UGAL, the parental wild type. Vg-DefA, transgenic mosquitoes with the Vg-DefA transgene; Vg-CefA, transgenic mosquitoes with the Vg-CefA transgene.

View larger version (26K): [in a new window]   Fig. 3. Structure of the transformation vector for transgenic alteration of the IMD/Relish pathway and its expression in transgenic mosquitoes. (A) Schematic diagram of the pBac[3xP3-EGFP afm, Vg-Rel] transformation vector that was transformed into the Ae. aegypti germ line. Three alternative spliced transcripts of Aedes Relish and Rel structure are shown. Q/H-rich, glutamine/histidine-rich domain; S-rich, serine-rich domain; RHD, Rel homology region 1; IPT, Ig-like plexin transcription factor domain; NLS, nuclear localization signal; ANK, ankyrin domain; DD, death domain. (B) Marked susceptibility to bacterial infection of the transgenic mosquitoes. Survival rates (%) of the parental wild-type (UGAL) and transgenic blood-fed mosquitoes (RMID) at 24 h after the injection of E. cloacae are shown. None of the transgenic mosquitoes at 24 h post-blood meal (PBM) survived more than 24 h, presenting the immune-deficient phenotype. (C) Resistance recovery of immune-compromised mosquitoes overexpressing the Defensin gene. Female RMID transgenic mosquitoes were mated to male Vg-DefA transgenic mosquitoes, and their progeny were subjected to survival test with E. cloacae. (Reproduced from Shin et al., 2003, with permission.)

View larger version (16K): [in a new window]   Fig. 4. Genetically dominant phenotype of the Vg-Rel transgene. Female Vg-Rel transgenic (RMID) mosquitoes were mated to male wild-type (UGAL) mosquitoes, and their progeny were challenged with bacteria. These hybrid mosquitoes showed a marked susceptibility to live bacteria, E. cloacae and E. coli. Heat inactivation of E. cloacae was performed by incubating the bacterial suspension at 95°C for 30 min. (Reproduced from Shin et al., 2003, with permission.)

View larger version (30K): [in a new window]   Fig. 5. A schematic representation of dominant-negative action of the Vg-Rel transgene. During normal immune response (left), bacterial challenge leads to activation of IMD/Relish signaling pathway (details are omitted) that results in proteolytic cleavage of ankyrin inhibitory domain from Relish and its translocation to the nucleus. In the nucleus, Relish binds NF-B binding sites in anti-microbial peptide (AMP)-encoding genes and activates their expression. In RIMD transgenic mosquitoes (right), the blood meal activates overexpression of Rel that lacks the transactivation domain but has the DNA-binding domain and nuclear translocation signal. Upon nuclear translocation, Rel binds NF-B binding sites in AMP-encoding genes. In these mosquitoes challenged with bacteria, Rel prevents normal Relish from binding to NF-B binding sites in AMP-encoding genes and their subsequent activation, causing immune deficiency. In the Vg-Rel/wild-type hybrid mosquitoes, the Vg-Rel gene still results in overexpression of Rel, preventing transactivation of AMP-encoding genes and thus acting as a dominant-negative gene.

View larger version (24K): [in a new window]   Fig. 6. Domain structure comparison of mosquito and Drosophila Rel-related factors with mammalian p105 and p100. Q/H-rich, glutamineand histidine-rich region; Q-rich, glutamine-rich region; S-rich, serine-rich region; G-rich, glycine-rich region; RHD, Rel-homology domain 1; IPT, Ig-like plexin transcription factor domains (Relhomology domain 2); NLS, Nuclear localization signal; DD, death domain.

View larger version (24K): [in a new window]   Fig. 7. Strategies for generating heritable and inducible RNAi. (A) A strategy for generating transgenic mosquitoes utilizing a strong inducible promoter. The construct containing a strong inducible promoter fused with an inverted repeat of a target gene is placed in the piggyBac transposable vector pBac[3xP3-EGFP afm], and transgenic mosquitoes are generated using an established transformation technique. Upon activation of these transgenic mosquitoes, transcripts are generated which fold back to form double-stranded RNA in all cells or tissues where the inducible promoter is expressed. (B) An alternative strategy utilizing a binary GAL4/UAS system. The inverted repeat is placed downstream of the upstream activating sequence (UAS) promoter, and a transgenic strain utilizing the pBac[3xP3-EGFP afm] vector is generated. A tissue-/stage-specific inducible promoter controlling GAL4 is transformed using the pBac[3xP3-DsRed afm] vector. Crossing two strains with different color selectable makers allows easy selection of hybrids in the F1 generation. The F1 hybrids contain both GAL4 and UAS genes. Tissue-specific activation of the GAL4-UAS transgene results in production of hairpin-loop RNA and consequently of RNAi.