Figure 1.
Cellfectin II® enhances GFP mRNA in vivo expression in Ae. aegypti.
(a) Cellfectin II® significantly enhances GFP expression in Ae. aegypti. 200 ng pAc-GFP and the empty vector DNA were microinjected per mosquito. Total RNA was isolated from whole mosquitoes and the samples were decontaminated by Dnase I treatment. The GFP expression was determined by SYBR Green® QPCR, and normalized using Ae. aegypti actin (AAEL011197). Each dot represents 1 mosquito. The experiment was repeated twice with similar results. (b) Cellfectin II® does not influence mosquito survival. Each liposome reagent mixed with vector DNA was microinjected into the mosquito (200 ng DNA each mosquito). The mosquito survival was recorded at 6 hours after materials inoculation. Control mosquitoes were treated with PBS. The number at the top of bar represents the number of live mosquitoes/total mosquitoes in a treatment.
Figure 2.
Cellfectin II® enhances GFP protein expression in Ae. aegypti.
Cellfectin II® facilitates GFP expression in Ae. aegypti (a) and in An. gambiae (c). 200 ng plasmids were microinjected into mosquitoes. The GFP protein concentration in whole mosquito lyses was measured by ELISA. Data are expressed as the mean ± standard error from 3 independent experiments. Titration of the amount of plasmid DNA influences the GFP expression in Ae. aegypti (b) and in An. gambiae (d). 2-fold titrated plasmid DNA with Cellfectin II® was inoculated into mosquitoes. The treated mosquitoes were sacrificed at 6 days post microinjection and homogenated in tissue lysis buffer. The GFP protein level was determined by ELISA. Data are expressed as the mean ± standard error from 3 independent experiments.
Figure 3.
Silencing AeTEPs enhances DENV infection.
AeTEPs were silenced by dsRNA treatment. The mock group was treated with the same amount of GFP dsRNA. The DENV burden was examined at 6 days post-infection. 10 MID50 DENV was used to challenge mosquitoes. The viral load was determined by Taqman® QPCR, and normalized by Ae. aegypti actin. Each dot represents 1 mosquito. The Mann-Whitney test was used for statistic analysis. The horizontal lines depict the medians of result. The result shown was combined with 3 independent experiments.
Figure 4.
In vivo overexpression of AeTEP-1 impairs DENV infection.
(a) AeTEP-1 expression in S2 cells. pAc-AeTEP-1 expressed a ∼200 KD recombinant protein in drosophila S2 cells. AeTEP-1 was detected with a V5-HRP mAb. The mock control was the empty vector transfected S2 cells. (b) Cellfectin II® facilitates AeTEP-1 expression in Ae. aegypti at 3 days (i), 6 days (ii) and 12 days (iii) post microinjection. Controls are the mosquitoes inoculated with empty pAc vector or only pAc-AeTEP-1 plasmid DNA in medium. The mRNA level of AeTEP-1 was determined by SYBR Green® QPCR, and normalized using Ae. aegypti actin. The experiment was repeated 3 times with similar results. (c) Overexpression of AeTEP-1 significantly decreased DENV burden. The viral load was determined by Taqman® QPCR, and normalized by Ae. aegypti actin. The result shown was pooled from 3 independent experiments. (b and c) Each dot represents 1 mosquito. The Mann-Whitney test was used for statistical analysis.