Validation of novel promoter sequences derived from two endogenous ubiquitin genes in transgenic Aedes aegypti

Abstract

To date, only a limited number of promoter sequences have been described to drive transgene expression in the disease vector Aedes aegypti. We sought to increase this repertoire by characterizing the ability of upstream sequences derived from the Ae. aegypti Ub(L40) and polyubiquitin genes to drive the expression of marker proteins. Both genomic fragments were able to drive robust expression of luciferase in cultured mosquito cells. Following Mos1-transformation, the Ub(L40) promoter drove strong expression of a fluorescent marker in early larvae and in ovaries, while the polyubiquitin promoter drove robust EGFP expression in all stages of development, including constitutive expression throughout the midgut. These promoter fragments provide two new expression profiles for future Ae. aegypti genetic experiments.

Figure 1

Ub_L40 and PUb promoters are highly active in mosquito cells. Schematic representation of Ub_L40 (A) and PUb (B) gene structure in Ae. aegypti. Exons (E) and introns (I) are indicated, with the predicted mRNA, including 5′ and 3′ untranslated regions (UTRs) and open reading frame (ORF) are shown below each gene. Numbers indicate length in base pairs. Gray boxes indicate the 76 amino acid ubiquitin monomer. Bars above each gene represent putative promoter regions utilized in (C, D) and all further experiments. (C, D) Relative luciferase activity from IE-1, Ub_L40 and PUb promoters compared to a no promoter control plasmid (pGL3) at 48hrs post-transfection in C6/36 (C) and Aag2 cells (D). Each experiment (Exp) was performed in triplicate. Statistical significance (*) was determined by Students t-test. Ub_L40 and PUb promoter/gene sequences have been deposited in Genbank (accession #s GU179017 and GU179018).

Figure 2

Southern analysis of Ub_L40-EGFP and PUb-EGFP insertions in Mos1-transformed Ae. aegypti. (A) Schematic representation of hypothetical transgene insertions for each of the donor constructs used. Block arrows represent the right (R) and left (L) arms of the Mos1 transposon. Bar indicates the size of the entire insertion and the dashed line represents mosquito genomic DNA. Restriction enzyme sites EcoRI (E), SalI (S), XbaI (X) and NsiI (N) are indicated. (B) Genomic DNA from each of the families identified as DsRED⁺ was digested with the indicated enzymes and hybridized with a probe corresponding to the Mos1 arms as well as the DsRED-SV40 gene cassette. The recipient strain kh^w is included as a negative control for all hybridizations. Molecular weight markers are shown at left (in kbp).

Figure 3

Ub_L40 and PUb promoters drive EGFP expression in transformed Aedes aegypti. EGFP expression in L₁ larvae (A) and pupae (B) for PUb-EGFP #P5, kh^w and Ub_L40-EGFP #P17A. (C) White light, EGFP and DsRED images of adult female kh^w, Ub_L40-EGFP #18 and PUb-EGFP #P5.

Figure 4

Northern analysis of Ub_L40-EGFP and PUb-EGFP expression in transformed Ae. aegypti. (A) Northern blot of endogenous Ub_L40 (kh^w, Lvp) mRNA and EGFP mRNA in transgenic lines Ub_L40-EGFP #18 and #P17A. Blots were probed with either the L40 portion of the Ub_L40 ORF (last 159bp) along with its 3′UTR or for EGFP. (B) Northern blot of PUb and PUb-EGFP expression in kh^w and PUb-EGFP #P5 transgenic mosquitoes. A probe corresponding to the PUb 5′UTR was used for hybridization to each blot, with ribosomal RNA shown as a loading control below. Individual lanes include: first through fourth instar larvae (L1-L4), male pupae (♂P) female pupae (♀P), adult whole body (WB), head (H), thorax without salivary glands (T), salivary glands (S), midgut (M), sugar-fed ovary (Os), and ovaries at 4 days post blood meal (Ob).

Figure 5

Ub_L40 and PUb promoters drive robust transgene expression in Ae. aegypti ovary and midgut tissues. (A) White light and EGFP images of kh^w, Ub_L40-EGFP #18 and PUb-EGFP #P5 ovaries at 24 hr, 48 hr and 72 hrs post blood meal. Thick white arrow indicates concentrated EGFP expression in nurse cells, thin white arrows indicate undeveloped secondary and tertiary ovarioles. (B) White light and EGFP images of midgut tissue obtained from kh^w, Ub_L40-EGFP #18 and PUb-EGFP #P5 mosquitoes.