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. 2014 Feb 4;4:3954.
doi: 10.1038/srep03954.

Identification of Germline Transcriptional Regulatory Elements in Aedes Aegypti

Free PMC article

Identification of Germline Transcriptional Regulatory Elements in Aedes Aegypti

Omar S Akbari et al. Sci Rep. .
Free PMC article


The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UD(MEL), and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.


Figure 1
Figure 1. RNAseq expression analysis.
RPKM expression values for AAEL007097 (A), AAEL007584 (C), AAEL010097 (E), and AAEL000923 (G) were plotted across development. Samples include, from left to right: testis; male carcasses (lacking testes); carcasses of females prior to blood feeding (NBF), and at 12 hr, 24 hr, 36 hr, 48 hr, and 72 hr; ovaries from NBF females and at 12 hr, 24 hr, 36 hr, 48 hr, and 72 hr; embryos from 0–2 hrs through 72–76 hr; whole larvae from 1st instar, 2nd instar, 3rd instar and 4th instar; male pupae; female pupae. Genome browser snap shots were extracted from the genome browser at for germline samples including both ovaries and testes (B,D,F,H,). Red box indicates coding sequence; green box indicates the annotated UTRs; yellow box depicts the putative regulatory regions chosen; and purple box indicates the SV40 UTR. Y axis in panels (A,C,E,G) indicates normalized expression levels in RPKM; the Y axis in the genome browser snapshots shows expression level based on raw read counts.
Figure 2
Figure 2. Fluorescent analysis of regulatory regions.
Previtelogenic NBF ovaries were imaged for each promoter:eGFP transgenic line to capture the overall expression pattern of follicles within an ovary (A top panel). The inset shows expression of the same ovaries at a higher magnification (A bottom panel). Individual ovarian follicles were imaged a 24 hr post blood meal (PBM) (B) and 72 hr PBM (C left panel) for each regulatory element. At 72 hr PBM a constant exposure was also taken of each oocyte (C right panel) so as to be able to compare relative levels of eGFP expression for different constructs. eGFP expression is also observed in the testes of AAEL010097 (D). Terminology: FE- follicular epithelium, NC- nurse cell, 2°F –secondary follicle, G –germarium, HC –hub cells, GSC –germ stem cells, SSC – somatic stem cells, MC –maturing cyst, and VE –vas efferens.
Figure 3
Figure 3. Germline specificity of regulatory regions.
Quantitative RT PCR for each Promoter:eGFP transgenic line measuring relative expression in the NBF ovary (red), NBF female carcass (dark green), 24 hr ovary (pink), 24 hr female carcass (green), testis (blue), and male carcass (light blue). Y axis is relative expression level. Error bars show standard error. To determine statistical significance, P-values were calculated between germline tissues and respective carcasses (brackets). P < 0.05 is indicated with one asterisk, p < 0.005 is indicated with two asterisks, p < 0.0005 is indicated with three asterisks.

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