Fly stocks and injections
Wild type Drosophila melanogaster (CS/S) flies and Df(1)RK4 mutant flies (Bloomington Drosophila Stock Center, Donors: Robert Kreber and Rachel Drysdale) carrying a deficiency of the X chromosome region 13A9-B1 were cultured at 25°C, 50–60% humidity, 12 h/12 h light/dark cycle, on yeast, dark corn syrup, and agar food. For the injections, flies were anesthetized whit CO2 (at a maximum of 5 min) [6, 7]. Using custom-beveled glass pipettes (20 × 40 μm tip diameter) coupled to a cell injector (Narashige IM-200) and a micromanipulator under a stereo microscope (Leica GZ6), we injected a volume of 0.2 μl/fly by a pulse pressure of 300 kPa [6, 7]. Injection of embryos was performed using the Nanoliter 2000 injector (World Precision Instruments).
dsRNA synthesis
For RNAi studies, we targeted two 22-nucleotide long regions of the blt gene; i.e., regions 2–23 and 685–708. Eight 39-mer DNA oligonucleotides with an attached T7 RNA polymerase promoter sequence were synthesized (Integrated DNA Technology, Inc.); these oligonucleotides represented both sense and antisense strands. The sequences of the sense and antisense DNA oligonucleotides corresponding to the blt gene used for the in vitro transcription reaction are shown in Table 1; only one match was found in the genome database pattern search analyses for each chosen targeted sequence.
One set of oligonucleotides produced dsRNA probes with a 3'overhanging 2 UUnt, and a second set was used for the production of blunt-end dsRNAs with GG..CC flanking nucleotides. Equal amounts of oligonucleotides were annealed to form a double-stranded template by heating at 80–85°C for 5 min and cooling down on ice. The in vitro transcription reaction (30 μl volume) for the synthesis of the 22 nt RNA run of transcripts contained 0.1 μg of a template, 500 μM each CTP, GTP, ATP, and UTP, 1 × transcription buffer (Tris-HCl, pH 7.5, 10 mM dithiothreitol, 1% bovine serum albumin), 20 U of RNAse inhibitor, and 50 U T7 RNA polymerase (Gibco BRL). The reactions were carried out at 37°C for 1 h. The RNA molecules were annealed together in heat denaturing conditions (65°-70°C for 5 min) and placed on ice. The quality of both RNA and DNA oligonucleotides was analyzed on 6% NuSive agarose gel (Sigma). As a negative control, we used dsRNA that targeted the human 5-lipoxygenase gene because Drosophila does not have any lipoxygenase homologues. The following is the cDNA sequence of a 22 nt of the human 5-lypoxygenase gene template that we used for in vitro dsRNA synthesis: 5'-ttcatgcacatgttccagtctt-3'; no match was found in the Drosophila genome database pattern search analyses.
To find an effective concentration of dsRNA for the RNAi studies, we tested four different dsRNA concentrations in pilot experiments; dsRNA was injected intra-abdominally into the males and females: 1 μg/μl, 0.5 μg/μl, 0.1 μg/μl, and 10 ng/μl in injection buffer (0.1 mM sodium phosphate, pH6.8, 5 mM KCl)(16.8–0.16 pmol, respectively). The most effective and longest lasting (5 days) silencing effect was detected with the lowest concentration of dsRNA (i.e., 10 ng/μl) for both probes, and we used this concentration in all subsequent experiments, except one experiment that was conducted with 100 ng/μl (where indicated).
Reverse transcription-polymerase chain reaction (RT-PCR)
To determine the presence of endogenous blt mRNAs and the effect of RNAi, we used an RT-PCR assay [6]. The total RNA was extracted from samples collected at different stages of Drosophila development. For adult flies, the total RNA was extracted separately from the heads and bodies of male and female flies; two flies were used for each experiment. Two embryos displaying cuticular defects and two normal embryos from Df(1)RK4 flies were used for RT-PCR. RNAs were DNAase treated prior to RT reaction. The rp49 gene was used as an internal control. The following are the primers used in the RT-PCR assay: for blt – direct 5'-atcgatacggaactctttgcgt-3', reverse 5'-atttcgctggggatcactaaac-3'; for rp49 – direct 5'-atgaccatccgcccagcataca-3', reverse 5'-tgtgtattccgaccaggttac-3'. The reaction conditions were as follows: first strand synthesis, 1 h at 37°C in 20 μl of reaction mix with a 0.2 mM of each dNTPs, 10 pmol of a hexanucleotide primers, 20 U RNAse inhibitor, and 100 U reverse transcriptase. We performed 30 cycles of PCR in 20 μl of reaction mix at the following conditions: 92°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec. Agarose gel electrophoresis was used to resolve and visualize the PCR reaction products.
Northern blot assay
Total RNA was extracted from the heads and bodies of male and female flies with a Trizol reagent (Gibco BRL). Ten micrograms of total RNA per sample were analyzed according to a standard protocol for the Northern blot analysis [35]. A digoxigenin-labeled 524 bp PCR fragment of blt was used as the hybridization probe. The signal was detected via chemiluminescence with the CSPD reagent (a substrate for alkaline phosphatase; Roche, catalog number 1655884); the blots were exposed for 12 h to Kodak BioMax film.
In situ hybridization assay of blt mRNA
The heads of 20–30 male and female flies were collected 72 h after injection with dsRNA oligonucleotides or ringer solution, and heads from naïve flies were also collected. The heads were sliced on dry ice with a sterile surgical blade (#11) and transferred into the tubes with a fixation buffer (15 % formaldehyde in phosphate-buffered saline; PBS; pH 7.4) for 20 min. Ovarian, corpus allatum, and ring gland hybridization was done on cryostat sections of female flies and 3rd instar larvae. For the 3rd instar larval muscle preparation, larvae were dissected from the ventral side with a surgical blade (#11), the cuticle was stretched and pinned down, and the preparations were fixed. The ovaries also were collected from anesthetized flies for confocal microscopy (Leica TCS-NT 1.6.587) without the in situ hybridization. They were manually dissected in PBS and fixed in a 1% glutaraldehyde in PBS buffer for 20 min, rinsed in PBS, and mounted in a NaCl/glycerol solution.
The in situ hybridization procedure was applied as described elsewhere [36]. Briefly, the samples were incubated with proteinase K (10 μg/ml) for 5 min at room temperature, rinsed with a glycine (2 mg/ml) in PBS, and prehybridized (6x SSC, i.e., 3 M NaCl, 0.3 M sodium citrate, 10 μg/ml sonicated salmon sperm DNA, 10 μg/ml yeast tRNA, 50 % formamide, 0.01 % Tween-20) at 55°C for 1 h. The samples were hybridized overnight at 55°C with digoxigenin-UTP-labeled (Roche) sense and antisense strands of a 524 bp fragment of blt gene (sequence 128–652; AF 173374). They were washed three times in PBS, 1 h each at 55°C, and incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (1:1000 in PBS at room temperature for 1 h). Thereafter, sections were washed 4 times 30 min each in PBS at room temperature, and rinsed twice in a blue color development buffer for alkaline phosphatase (100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20). The color was developed with NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; Roche) and the reaction was stopped with 2 mg/ml glycine in PBS, pH 2.2. Samples were mounted in a mounting solution (80% glycerol, 0.25 mM NaCl) and images were taken under the microscope.
Since we were not able to detect mRNA staining in embryos with conventional in situ hybridization (probably because the signal is below detection level), we used an in situ RT-PCR assay with digoxigenin-labeled dUTP to localize bl t mRNA in the embryos. Briefly, the 0–20 h embryos were collected on agar plates and fixed and processed as described elsewhere [36]. The RT step was carried out with at least 200 embryos per tube and in 20 μl of digoxigenin-labeling PCR mix (Roche); the samples were processed for 10 cycles (92°C 15 sec, 57°C 30 sec, 72°C 30 sec). Thereafter, they were washed several times in PBT (PBS/Tween-20), incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (1:1000 in PBS) for 30 min, and washed in PBS for 2 h. Specific primers used in this PCR reaction were as follows: direct-5'-tagtcggctgatgaggattgttcc-3', reverse-5'-acacgtagttgatgtcacccatgg-3'. As a negative control, either one or both primers were omitted from the above-described procedure.
Cuticle preparation
Embryos were manually dechorionated, covered with mineral oil on a glass slide, and flattened with a cover slip. They were steam-fixed on top of boiling water for 1 min, the cover slips were slowly removed and the mineral oil was replaced with a mounting solution (glycerol/NaCl). The vitteline membrane and all debris were manually removed and covered with a cover slip. The preparations were examined under a microscope and photographed.
Oviposition
Mated female flies were injected with 10 ng/μl of blt dsRNA, control dsRNA, or with buffer and individually placed for 24 h into small glass tubes with food. Flies were transferred to new tubes every 24 hours. The tubes were inspected under the microscope and the number of eggs laid was counted.
