Arginase knockdown in Aedes aegypti

A gene encoding urate oxidase (UO, VectorBase AAEL002194) was previously studied in A. aegypti[13] (link). In this report, a putative ortholog of the mosquito arginase gene (AR, VectorBase AAEL002675) was identified by BLAST searches using fruit fly arginase (FBpp0070083-PA as a query [21] (link)). The arginase gene in A. aegypti is a single copy gene. It encodes a protein of 349 residues and shares 85%, 43% and 54% identity to Anopheles gambiae, D. melanogaster, and Bombyx mori, respectively.
UO, AR and firefly luciferase (FL, GenBank accession number U47295) gene-specific primers flanked with T7 promoter sequence (Table 1) were used to PCR amplify DNA from mosquito cDNA and pGL3 vector (Promega, Madison, WI). Double-stranded RNA (dsRNA) covers approximately the first half of the coding sequence of UO or AR genes, and the target region corresponds to the catalytic domain of each protein. The dsRNA was prepared as described previously [17] (link). Newly-eclosed females were injected with 500 ng of dsRNA using a Nanoject II microinjector (Drummond Scientific Company, Broomall, PA) [13] (link). Females were fed with a blood meal 4 days later.
The FB and MT were dissected from individual mosquitoes at 24 h and 48 h after blood feeding. Total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA) and reverse transcribed using oligo-(dT)₂₀ primer and reverse transcriptase (Promega, Madison, WI). The cDNA was then used as a template for qRT-PCR assays using gene-specific primers (Table 1). UO and AR knockdown efficiency, as well as the relative mRNA level of several other genes involved in nitrogen metabolism in A. aegypti were analyzed by qRT-PCR. Briefly, qRT-PCR was performed with PerfeCTa SYBR Green FastMix, ROX (Quanta BioSciences, Gaithersburg, MD) and a final primer concentration of 200 nM using Applied Biosystems 7300 Real-Time PCR System (Life Technologies, Carlsbad, CA) and the following PCR conditions: 95°C for 2 minutes followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. The PCR efficiency of a primer set for each gene were verified by performing a dilution series experiment with a corresponding cloned plasmid DNA. Relative level of expression for each gene was calculated using the ΔΔC_T quantification method [24] (link). Ribosomal protein S7 transcript levels were used as an internal control for normalization of mRNA yields in all samples. The knockdown efficiency was determined as previously described [13] (link).