A BLAST search of the Ae. aegypti
genome (genebuild AaegL1.1) was performed based on the 30K a
sequence on the VectorBase website (http://www.vectorbase.org
) (Altschul et al., 1990
). This search revealed that these genes are located on the supercontig 1.464. A third member of the family also was found on the same supercontig.
All oligonucleotide primer sequences for gene constructions and primary sequence validation are listed in Supplemental Table 1
. A schematic representation of the final constructs utilized to generate transgenic mosquitoes is shown in . The 30K
putative promoter was amplified using genomic DNA extracted from Higgs mosquitoes as template and primers 30 K P FP and 30 K P RP that anneal in the first exon of both genes. The generated amplicon was cloned into the pGlow-topo plasmid (Invitrogen) at the 5’-end of the EGFP ORF and the BGH polyadenylation signal. The SV40 signal was amplified from pDsRed-monomer-N1 (Clontech) with primers SpeIsv40 FP and SpeIsv40 RP and cloned at the 3’-end of the 30K a
gene in the Spe
I site. The construct was amplified from pGlow-topo with primers AscI SV40 F and EcoRV BGH R and cloned into blunted Fse
I and Asc
I sites in the pMos3XP3DsRed plasmid (Horn et al, 2002
) to generate pMos30K.
30KExGM and GFP30KMnp were constructed by initially cloning the Mnp
sequence from plasmid pMos-carb/Mnp/i/Mnp/svA (Franz et al., 2006
) into the Not
I and Bam
HI sites of the pSLfa plasmid (Horn et al., 2002
). The putative 30K
promoter with the 5’UTRs for both 30K a
genes and complete ORF for 30K a
gene was amplified from Higgs genomic DNA, and separate fragments with and without the first 30K b
exon were made using primer pairs 30K PstI for and 30K NotI rev exon and 30K PstI for and 30K NotI RP N, respectively. These amplified DNA fragments then were cloned to the 5’-end of Mnp
in pSLfa. A 54 bp linker sequence formed by the annealing of Not
I sense and antisense oligonucleotides also was cloned between the 30K b
first exon and Mnp
in the construct 30KExGM. EGFP ORF, 30K a
3’UTR and 30K b
3’UTR were amplified using primer pairs GFP SacII for and GFP PstI rev, 30Ka 250 3utr speI F and 30K a 3UTR sacII RP and 30KbutrBamHIF and 30Kb 3340 3utr EcorI R, respectively, and cloned into the pSLfa plasmid. The constructs were cloned into Asc
I site in the pMos3XP3DsRed plasmid.
Preblastoderm embryos from Aedes aegypti
Higgs white-eyed strain (Higgs et al., 1996
) were injected with the pMos30K, 30KExGM or GFP30KMnp constructs and Mos1 mariner
helper plasmids, following established protocols (Jasinskiene et. al. 1998
). Lines are maintained by intercrossing transgenic siblings.
Surviving G0 males were mated individually or in groups of three with 10-15 or 45 Higgs females, respectively. G0 females were mated in pools of 7-8 with equal numbers of Higgs males. G1 larvae were screened for red fluorescence in the eyes. Transformed animals were raised and mated with Higgs mosquitoes.
Southern blot analyses
Genomic DNA was extracted from six transgenic or Higgs wild-type females or fourteen transgenic males using the Promega wizard genomic DNA extraction kit. Genomic DNA was digested overnight with the restriction enzymes EcoRI or XhoI. DNA fragments were separated on a 1% agarose gel followed by overnight transfer to a BioRad Zeta probe nylon membrane. The blot was probed with a 700bp DsRed DNA fragment labeled with 32P. The probe was hybridized at 65°C overnight, the blot washed and exposed to x-ray film or a phosphor screen.
Northern blot analysis
Total RNA was extracted from the salivary glands of twelve transgenic females, carcasses (whole-body excluding salivary glands) of six transgenic females, six whole transgenic males, six whole Higgs females or six whole Vg40 transgenic females using Trizol (Invitrogen). RNA was separated on a 1.2% formaldehyde gel for 3 hrs at 70V. The Millenium markers (Ambion) were used as molecular weight standards. The RNA was transferred to Zeta probe nylon membrane overnight. The blot was probed with EGFP or Mnp fragment labeled with 32P. The probes was hybridized at 65°C overnight, washed and exposed to a phosphor screen.
Total RNA was extracted from the heads, thoraces and abdomens of the transgenic females, whole transgenic males or whole Higgs females using Trizol (Invitrogen). 500ng of RNA from each sample were treated with DNAseI (Invitrogen) for 15 minutes at room temperature. The reaction was terminated by addition of 25mM EDTA followed by inactivation of DNAse at 65°C for 10 minutes. One-step RT-PCR was performed using the Qiagen One step RT-PCR kit and 100ng of DNAse-treated RNA as template. Primers AscI SV40 F and 30KA ORF F were used to amplify the 30K a
transgene at an annealing temperature of 69°C, and primers EcoRV BGH R and 30KB ORF F were used to amplify transgene 30K b
. Actin FP and RP were used to amplify the Aedes actin1
gene sequences (Ibrahim et al., 1996
). The annealing temperature of 62°C was used to amplify 30K b
and the actin control.
Salivary glands were dissected from 15 transgenic or Higgs mosquitoes and homogenized in 15μL of lysis buffer, which is prepared by dissolving complete mini (Roche) protease inhibitor cocktail (serine, cysteine and metalloprotease inhibitors) and Peflabloc SC (Roche) in water. Two carcasses reserved after the dissection of salivary glands and two males from transgenic or Higgs females were homogenized in 100μL of lysis buffer. An equal volume of Laemlli buffer (BioRad) with 0.1M dithiothreitol (DTT) was added to all samples. Samples were heated at 95°C for 5 min and centrifuged for 5 minutes before loading on a 12% Tris-HCl Ready gel (BioRad). 10μL of BioRad Dual color precision marker were used to calculate the Mr of proteins. Following electrophoresis, proteins were transferred to PVDF membrane using semidry transfer system for 1.5 hr at 33V. Proteins were stained with Coomassie Stain solution (BioRad) and the membrane blocked overnight in 5% nonfat dry milk at 4°C. The membrane was incubated in 1:1000 anti-GFP antibody (Santa Cruz) for 1 hour at room temperature followed by incubation in 1:50,000 alkaline phosphate-conjugated goat anti-rabbit IgG (Jackson Immunosearch). Alkaline phosphatase activity was detected by incubating the membrane in NBT/BCIP (Roche) substrate solution for 30 minutes. All washes and antibody dilutions were made in 1X TBS (150 mM NaCl, 100 mM Tris) with 1% Tween-20.
Salivary glands were dissected from 5-day old transgenic or Higgs wild-type females in 1 X PBS and examined for EGFP fluorescence under a dissecting microscope fitted with UV optics.
Detection of siRNA
Total RNA was extracted from whole mosquitoes, males or females, dissected salivary glands or carcasses (whole body excluding salivary glands) using Trizol (Invitrogen). RNA samples were submitted to electrophoresis on a 15% TBE-urea polyacrylamide gel. After electrophoresis, RNA was transferred to a neutral Hybond Nx membrane for one hour at 20V at 4°C and crosslinked using carbodiimide for 1.5 hrs at 60°C (Pall et al., 2007
). The membrane was incubated in UltraHyb hybridization buffer (Ambion) with salmon sperm DNA at 42°C for 1 hour. The 500nt sense fragment of Mnp
with T7 promoter on the 3’end was used to prepare in vitro
P labeled RNA probe (MEGAscript kit, Ambion). A total of 7μg of labeled probe was hydrolyzed using carbonate buffer for 2.5 hrs and added to the prehybridized blot in UltraHyb buffer for overnight incubation at 42°C. The membrane was washed in 2 X SSC, 0.1% SDS twice for five minutes, in 0.1 X SSC, 0.1% SDS twice for fifteen minutes each time at 55°C, and exposed to the phosphor screen for image detection. A 22nt commercially-synthesized (Sigma-Aldrich) RNA oligonucleotide 5’AUUUAACCACACGUAAUGGAGA 3’ corresponding to a segment of the sense-strand of Mnp
was used as the positive control.
Cell and virus culture
Monkey kidney cells (LLC-MK2; ATCC CCl-7.1) and Aedes albopictus C6/36 cells were obtained from the American Tissue Culture Collection (Prince William County, Virginia). Vertebrate and insect cells were cultured in Modified Eagle's medium (MEM) supplemented with 8% heat inactivated (56°C for 30 minutes) fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% glutamine, 1% non-essential amino acids and maintained at 37° C and 28° C, respectively, in an incubator with 5% CO2. High-passage DENV-2 stock (JAM 1409) was used to propagate virus in C6/36 cells. Briefly, monolayers of C6/36 cells were inoculated with DENV-2 at a MOI of 0.01 and held at 28° C. The supernatant was collected on the day with the highest cytopathic effect (6-8 days post-inoculation). Concentrations of DENV-2 particles were determined by plaque assay.
DENV2 challenge by the blood feeding route
Seven to eight days after eclosion, female mosquitoes (100–150/one-pint carton) were starved of sucrose and deprived of water for 24 hours prior to blood feeding. The mosquitoes were challenged with an artificial infectious blood meal consisting of 40% (v/v ) defibrinated sheep blood (Colorado Serum Co., Boulder, CO), DENV2 Jamaica 1409-infected C6/36 cell suspension (60 % v/v), and 1 mM ATP. Blood meals were maintained at a constant temperature of 37°C. Mosquitoes were allowed 1 hour to feed. Blood meals were titered post-feed to verify virus concentration. Fully-engorged mosquitoes were selected and held for 14 days at a constant temperature of 28°C and a relative humidity of 80% in an insectary with a 12-hour photoperiod.
DENV-2 transmission assay
Fourteen days after receiving a DENV2-containing blood meal, females were put in small cartons in groups of 7-8 mosquitoes. Sugar and water were removed one day before the assay. Females were allowed to probe and feed on 350 μl of a feeding solution (50 % FBS, 50 % MEM, 0.2 mM ATP, ~50 μg sucrose/phenol red, pH 7) that was placed between two parafilm membranes stretched over a glass feeder. After probing, mosquitoes and feeding solutions were collected and plaque assays were performed to determine virus titer.
The method for intrathoracic inoculation of mosquitoes is derived from Gubler and Rosen (1976)
. Adults female mosquitoes (5 days post-eclosion) were infected by injecting 69 nL of DENV-2 Jamaica 1409 (100 or 10 pfu) using a Nanojet II (Drummond Scientific Company, Broomall, PA). All injections were performed under a dissecting microscope using glass needles prepared with a vertical pipette puller (P-30, Sutter Instrument Co., Novato, CA). Mosquitoes were incubated for 7 days at 28°C, 80% relative humidity. The salivary glands were dissected and triturated in 0.5 ml of MEM medium. After centrifugation, the supernatant fluid was filtered (Acrodisc Syringe filters with 0.2 μm HT Tuffryn membrane) and virus titer determined by plaque assay.
LLC-MK2 cells were grown in confluent monolayers in 24-well plates. Ten-fold serial dilutions of salivary gland or whole-mosquito supernatants were added for 1 hour and cells were overlaid with agar. After 7 days of incubations at 37°C cells were stained with a solution of 3mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) added directly to the plate and incubated for 4 hours (Sladowski et al., 1993
, Takeuchi et al., 1991
). Viral titers were determined by plaque counting.
To determine viral titer and prevalence of DENV-2 infection in carcass and salivary glands, data were subjected to analysis of variance (ANOVA) and Pearson chi-square test, respectively, by using SAS (SAS User's Guide, Cary, NC: Statistical Analysis System Institute, Inc., 1987). Sources of variation were mosquito lines (transgenic lines; 30KExGM [1, 15, 22, and P4] and GFP30KMnp P6, and as controls Higgs and pMos-30K strains), replicate (two) and their interaction. For the intrathoracic injections, the two viral concentrations tested did not differ in the magnitude of viral titers in either carcasses or salivary glands, and no significant interaction between viral concentration and mosquito lines was detected; therefore they were considered as replicates. Similarly, the differences in viral prevalence between transgenic and control mosquitoes was identical between viral concentrations. Due to the lack of virus in saliva in most transgenic mosquitoes in the transmission assay, the averages of the five transgenic lines were pooled for comparison with the control group. If variances were not homogeneous, data were subjected to log-10 transformations. When differences among treatment means were detected, they were separated using least significant difference (LSD) procedure adjusted by Tukey-Kramer.