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 UbL40 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 UbL40 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.

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

“…aegypti genome, ensuring that no highly similar left and right binding sites were in close proximity. Donor vectors for HDR-based transgene insertion were generated using standard cloning methods by inserting amplicons derived from the respective target regions upstream and downstream of the PUb-EGFP cassette in the previously described vector pSLfa-PUb-EGFP (49). TALEN target site sequences and detailed assembly methods of both TALENs and donor vectors are provided in SI Appendix.…”

Genetic technologies for disease vectors

“…aegypti [37], the immediate early-1 gene from a baculovirus [38], the Actin 5C gene from D. melanogaster [39], and the genes ubiquitin (Ub(L40)) and polyubiquitin from Ae. aegypti [40].…”

Live calcium imaging ofAedes aegyptineuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies

“…aegypti strain harboring genomic sources of a genetically-encoded calcium indicator, GCaMP6s [30] . To express GCaMP6s, we utilized the polyubiquitin promoter (AAEL003877, henceforth PUb ), chosen for its generally high expression during nearly all developmental life stages and tissues as shown by previous promoter characterization experiments and developmental transcriptional profiling ( Figure 1A) [31,32] . We inserted the PUb promoter upstream of the coding sequence for GCaMP6s within a randomly inserting piggybac transposable element.…”

“…The predicted Ae. aegypti polyubiquitin ( PUb ) promoter fragment [73] was amplified from Ae. aegypti genomic DNA using primers 997.C1 and 997.C2.…”

“…http://dx.doi.org/10.1101/345389 doi: bioRxiv preprint first posted online Jun. 12, 2018; chosen for its generally high expression during nearly all developmental life stages and tissues as shown by previous promoter characterization experiments and developmental transcriptional profiling ( Figure 1A) [31,32] . We inserted the PUb promoter upstream of the coding sequence for GCaMP6s within a randomly inserting piggybac transposable element.…”