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. 2011 Aug;41(8):637-44.
doi: 10.1016/j.ibmb.2011.04.004. Epub 2011 Apr 27.

Targeted gene expression in the transgenic Aedes aegypti using the binary Gal4-UAS system

Vladimir A Kokoza  1 , Alexander S Raikhel
Affiliations

Targeted gene expression in the transgenic Aedes aegypti using the binary Gal4-UAS system

Vladimir A Kokoza et al. Insect Biochem Mol Biol. 2011 Aug.

Abstract

In this study, we report the establishment of the binary Gal4/UAS system for the yellow fever mosquito Aedes aegypti. We utilized the 1.8-kb 5' upstream region of the vitellogenin gene (Vg) to genetically engineer mosquito lines with the Vg-Gal4 activator and established UAS-EGFP responder transgenic mosquito lines to evaluate the binary Gal4/UAS system. The results show that the Vg-Gal4 driver leads to a high level of tissue-, stage- and sex-specific expression of the EGFP reporter in the fat body of Vg-Gal4/UAS-EGFP hybrids after blood-meal activation. In addition, the applicability of this system to study hormonal regulation of gene expression was demonstrated in in vitro organ culture experiments in which the EGFP reporter was highly activated in isolated fat bodies of previtellogenic Vg-Gal4/UAS-EGFP females incubated in the presence of 20-hydroxyecdysone (20E). Hence, this study has opened the door for further refinement of genetic tools in mosquitoes.

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Figures

Fig. 1
Fig. 1
Schematic representation of two constructs, the pBac[3xP3-EGFP afm, Vg-Gal4] – driver (A) and the pBac[3xp3-DsRed af, UAS-EGFP] – responder (B) used in germ-line transformation experiments. The 2.8-kb driver transgene consists of the Ae. aegypti mosquito’s Vg- 5’ promoter region linked to yeast Gal4 activator sequence with DNA binding domain (amino acids, 1–93) and activation domain (amino acids, 753–881), followed by SV40 polyA signal (A). The 1.5-kb responder transgene contains the UAS sequence with 5× concatamers of the Gal4 binding sites fused to the EGFP reporter gene with SV40 polyA signal (B). The transgene-specific primers used for genomic PCR, RT-PCR and inverse PCR are indicated by the arrows above and below the diagram.
Fig. 2
Fig. 2
Stable incorporation and integrity of the Vg-Gal4 driver and UAS-EGFP responder transposons into the Ae. aegypti genome. Genomic DNA was isolated from transgenic mosquitoes of the Vg-Gal4/UAS-EGFP hybrids (A), the driver line (B), the responder line (C), and non-transgenic UGAL strain (D). PCR amplification was performed using a set of primers specific to the left and right arms of the piggyBac vector (L arm and R arm), the Vg-Gal4 transgene (Gal4) and the UAS-EGFP transgene (UAS). Primers to Ae. aegypti actin were used to confirm the integrity of genomic DNA.
Fig. 3
Fig. 3
Blood meal activation of the binary Vg-Gal4/UAS-EGFP expression system in the fat body of the transgenic Ae. aegypti mosquitoes. RT-PCR analysis was performed using RNA samples isolated from the fat body of transgenic (A, B, C) and wild-type UGAL (D) mosquitoes. Expression profiles of the Vg-Gal4, UAS-EGFP transgenes, vitellogenin and actin in previtellogenic (PV) and vitellogenic females were analyzed 12, 24, and 48 h post-blood meal (PBM) using gene-specific primers.
Fig. 4
Fig. 4
Tissue- and sex-specific expression of the reporter gene in the Vg-Gal4/UAS-EGFP hybrid mosquitoes. Fat bodies (FB), ovaries (OV), midgut (MG), and Malpighian tubules (MT) from vitellogenic Vg-Gal4/UAS-EGFP hybrid female mosquitoes, 24 h post-blood meal (PBM), were analyzed by means of RT-PCR analysis. Specific amplification of the EGFP reporter was detected only in vitellogenic fat bodies. Males (M) were also negative for EGFP reporter RNA. Actin-specific primers were used as a control for RNA integrity and loading.
Fig. 5
Fig. 5
Tissue- and stage-specific EGFP reporter expression in the Vg-Gal4/UAS-EGFP hybrid female mosquitoes after blood meal activation. Fluorescent images of adult vitellogenic females were captured using an EGFP filter set. Expression of EGFP reporter was detected only in the fat body of hybrid Vg-Gal4/UAS-EGFP females 24 (A) and 48 (B) h post-blood meal (PBM). No EGFP fluorescence was observed in the fat body of vitellogenic (24 h PBM) females of the Vg-Gal4 driver (C) and UAS-EGFP responder (D) transgenics, or non-transgenic UGAL strain (E).
Fig. 6
Fig. 6
20E hormonal activation of reporter mRNA expression in the Vg-Gal4/UAS-EGFP hybrids in previtellogenic fat body in an in vitro organ culture. Previtellogenic fat bodies were incubated in culture media in the presence (20E+) or absence of (20E−) of 20E. RT-PCR analysis was performed using gene-specific primers to UAS-EGFP reporter, Vg-Gal4 activator and actin as a loading control.
Fig. 7
Fig. 7
Fluorescent images of previtellogenic fat body of Vg-Gal4/UAS-EGFP hybrids after 20E hormonal activation in an in vitro organ culture. Fat bodies dissected from previtellogenic hybrid transgenic female mosquitoes were incubated in the culture medium in the presence of 20E and prepared for examination by fluorescence microscopy. The tissue was stained by Hoescht DNA staining for visualization of nuclei. Imaging was performed under a Zeiss AxioObserver A1 microscope using EGFP filter (A), Blue filter for nuclei staining (B), and both filters to obtain a merged image (C). All images have a 50-µm scale.
Fig. 8
Fig. 8
Comparison of EGFP reporter expression in fat bodies of transgenic and non-transgenic female mosquitoes after 20E activation in an in vitro organ culture. Specific EGFP expression was observed only in the Vg-Gal4/UAS-EGFP hybrids (A), but not in the Vg-Gal4 driver (B), UAS-EGFP reporter (C), or non-transgenic UGAL females (D). Imaging was performed with a Zeiss AxioObserver A1 microscope using EGFP and Blue filters to obtain merged images of fat body preparations.
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