Insect rearing and germline transformation

The CA06 and MD wild type strains of L. sericata were raised under similar conditions as described previously [48, 70]. To make the MDLA mixed strain, 16 MD males were mated with 30 CA06 virgin females, the offspring collected and reared to adults. For heat shock experiments, MDLA or L. sericata PD1 eggs were placed on MYEA (50 g whole egg powder, 25 g instant non-fat milk powder, 12.5 g inactivated dry yeast, 7.5 g agar, 500 mL deionized water) at 27 °C overnight. First instar larvae were transferred to a new MYEA dish then heat shocked at 37 °C or left at 27 °C control for 3 h. The larvae were collected and then washed with water using a filter, attached to an aspirator. Larvae were then snap frozen in liquid nitrogen and stored at -80 °C. Introgression of the DR4#14 line into a L. sericata genetic background was done by crossing 125 DR4#14 L. cuprina males with 80 MDLA virgin females. Male offspring were then crossed with MDLA virgin females for two generations. The male and female offspring allowed to breed freely to establish the line.

piggyBac-mediated germ-line transformation of L. sericata was as previously described [47, 48] using a mixture of Lchsp83-pBac DNA (200 μg/mL) and in vitro synthesized piggyBac RNA helper (300 μg/mL). Homozygous Lucilia individuals were selected at the wandering third instar larval stage based on fluorescence intensity and bred to create a stable line.

Plasmid construction

To make the pB[Lchsp24-pdgf-B] construct, firstly the Lchsp24 promoter [45] was amplified from L. cuprina genomic DNA with the primer pair 5'-GAGCTCCTCGAGTAGGGTGGGCAATTTTTTCTAATGCCCATTA-3' and 5'-GGATCCTGGATAGGCTTCACGGTCCAGTTCATCGAT-3' and the fragment inserted into pBAC2 vector [47]. The Lchsp70 3' UTR and 3' flanking DNA [45] were amplified from L. cuprina genomic DNA with the primer pair 5'-AAGCTTAGTCAATCTCAATTTCATTCC-3' and 5'-GCGGCCGCCTCGAGAATGATATATACAAGGA-3', and inserted downstream of the Lchsp24 promoter. However, the Lchsp24 promoter fragment retained the start codon, so this was removed by amplification of a portion of Lchsp24 without the start codon with the primer pair 5'-ATTATCATTATCTACTAGTTCAGTTCTAGTTAC-3' and 5'-ATTATCGGATCCCTCTTTGGTTTTCTTAAA ACG -3', then digested with SpeI and BamHI(blunt) and inserted into SpeI and AgeI(blunt) of pB[Lchsp24-pl-Lchsp70], effectively replacing the promoter with an identical fragment minus the ATG codon. A DNA fragment was synthesized by Genscript that encoded the human PDGF-B protein and a 20 amino acid signal peptide (MKSFLLVLFAFLAVFAFVQA) from an 87 amino acid venom peptide that was expressed in L. sericata salivary glands [46] and detected in larval ES (P.H. Nibbering, personal communication). The nucleotide sequence was optimized for expression in L. cuprina. The pdgf-B Genscript plasmid was digested with flanking Asp718 (blunt) and AvrII and inserted into NheI (blunt) and AvrII of pBAC2[Lchsp24-Lchsp70]. A PspOMI and partial SpeI digest of pB[Lchsp83-ZsGreen-tub.2] [47] released a 3.3 kb Lchsp83-ZsGreen-tub.2 cassette which was inserted into PspOMI and SpeI of pBSII KS+. The resulting pBSII KS+[Lchsp83-ZsGreen-tub.2] plasmid was digested with NotI and PspOMI and inserted into NotI digested pB[Lchsp24- pdgfB -Lchsp70] to create the final piggyBac transformation vector, pB[Lchsp24-pdgf-B].

To construct DR4 and EF-PDGF, the general strategy was to first assemble the driver or effector gene cassette in the cloning vector pBluescript II KS (-) and then excise the gene cassette by digestion with XhoI and NotI and clone into the unique XhoI and PspOMI sites in the piggyBac transformation vectors pB[Lchsp83-ZsGreen] [47] or pB[Lchsp83-DsRedex2] [48]. To assemble the effector construct, a fragment encoding PDGF-B with N terminal signal peptide was amplified from pB[Lchsp24-pdgf-B] using the primer pair 5'-TTATCATGAAGTCGTTCTTGTTGGTGTTG-3' and 5'-TGAAAGCTTAGGTCACGGGACGGGCGGCAG-3', digested with BspHI and HindIII, and ligated to pBS-FL11 (Li et al. [48]) that had been digested with NcoI and HindIII. The effector gene cassette was then excised from the pBS-EF-PDGF plasmid and ligated with the pB[Lchsp83-DsRedex2] transformation vector.

For 5' RACE, total RNA was extracted from C. hominivorax embryos using TRI reagent (Sigma). polyA+ RNA was purified by oligo-dT chromatography (Sigma). 5' RACE-ready cDNA was prepared using the SMARTer RACE cDNA amplification kit (Takara) according to the manufacturers instructions. RACE-ready cDNAs were diluted in 20 μL TE buffer (Clontech), and then 2 μL was used for 5' RACE with the primer 5'-CAATTCACGCAAGAAAATCTCTTTGTTGGAATAGAAGGT-3'. “Genome walker” libraries were prepared from C. hominivorax genomic DNA using the Universal GenomeWalker kit (Takara) following the manufacturers instructions. PCR was then performed using the Chhsp83 gene specific primers (GSP) 5'-GATCAACCACAATCTAATATATTATAACTTTTTTCACTTTTCAGTT-3' and 5'-TTGTCTTTTCGCTCGCTTGGAAACTCTCGATGTAT-3'. The Chhsp83 gene promoter fragment for the DR4 construct was obtained by PCR amplification with genomic DNA template using the primers 5'-ATAGCGGCCGCTGTCATTACTAGAGTTTAAGTTATAACAATTGTAT-3' and 5'-ACGCTGCAGATCTGGAAATACAATAGGAAAAATAAAGTTAGCGAATT-3', then cloned into pGEM-T (Promega). The Chhsp83 promoter fragment was excised and ligated with pBS-FL1 that had been digested with NotI and PstI, essentially replacing the tetO-Dmhsp70 enhancer-promoter with the Chhsp83 promoter. The Chhsp83-tTAv-SV40 gene cassette was then excised and ligated with the pB [Lchsp83-ZsGreen] transformation vector.

Genomic DNA isolation and inverse PCR

Five to 6 frozen adults were ground to powder with a mortar and pestle under liquid nitrogen. Powder was dissolved in 4 mL fresh STE buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM EDTA, pH 8). Two hundred μL 10 % SDS and 8 μL RNase A (Cat# R4642-250MG Sigma Aldrich St. Louis, Missouri) were added and samples were incubated at 56 °C. After 30 min, Proteinase K (Cat# P2308-100MG Sigma Aldrich) was added to 100 μg/mL and the sample was incubated overnight at 56 °C. Three mL phenol:chloroform:isoamyl alcohol [25:24:1] (Cat#P2069, Sigma) was added and samples were rotated 10 min at 12 RPM at 22 °C. Samples were then centrifuged 10 min at 1000 g at 4 °C. The aqueous layer was transferred to a new tube and the extraction was repeated. One tenth volume 3 M sodium acetate, pH 5.2 and 2 volumes cold 100 % ethanol were added and mixed. The samples were incubated at -20 °C for 1 h and centrifuged 30 min at 7200 g at 4 °C. The supernatant was removed from the pellet, which was washed with 1 mL cold 75 % ethanol. The pellet was air-dried 10 min before resuspension in 50–100 μL TE Buffer. To determine genomic sequence flanking the transgene insertion, inverse PCR was performed with MboI, TaqαI, and MspI-digested genomic DNA templates as described previously [48, 71].

RT-PCR and qRT-PCR

RT-PCR was performed with cDNA template from total RNA isolated from larvae as described previously [72]. The pdgf-b primer pair used were PDGF-F (5-' ATG AAG TCG TTC TTG TTG GTG TTG TTC GCC TTC TTG GCC GTT-3') and PDGF-R (5'- CCG GAG TTT AAA CCC TAG GCG CGC CAT GAG CTC AAG CTT TCA TTA-3'). For RNA isolation for quantitative RT-PCR (qRT-PCR), 5-6 frozen larvae were homogenized in 500 μL of Trizol (Cat#15596026 Life Technologies/ Thermo Fisher Scientific Waltham, Massachusetts) in a 1 mL glass homogenizer that had been previously baked at 200 °C overnight. 100 μL of chloroform was added, and samples were shaken for 15 s and allowed to incubate at 22 °C for 15 min. Samples were centrifuged at 18,000 g for 15 min at 4 °C. The aqueous layer was mixed with an equal volume cold RNase-free 70 % ethanol, mixed, and loaded on a Qiagen RNeasy Mini Kit column (Cat#74104 Qiagen Venlo, Netherlands). The purification was performed according to the kit protocol, including the optional on-column DNase digest using the RNase-free DNase set (Cat#79254 Qiagen). A subsequent in-solution DNase digest was performed to eliminate residual DNA, followed by a second round of column purification. cDNA was synthesized from 3.5 μg of DNase treated RNA using Superscript III (Cat#18080-400 Invitrogen) according to manufacturer's instructions. Random hexamers were used as primers. Negative control reactions containing water instead of enzyme mix were performed to confirm the absence of DNA contamination.

PDGF qPCR primers were designed using Primer3: hPDGF F (5'- GAAATTGTGCGTAAAAAGCCCATTT-3') and hPDGF R (5'- AACAGTTTCACATTTACAGGCCAAA-3'). Primers were tested for efficiency by creating a dilution series of cDNA. Template was pipetted into quadruplicate wells of a 384 well optical plate (Cat#4309849 Applied Biosystems). Thermo Maxima SYBR Green/ Rox qPCR Master Mix 2X (Cat#K0221 Thermo Fisher Scientific Waltham, Massachusetts) was added to primers to create a master mix, which was dispensed into wells via a multi-channel pipet. The plate was sealed (Cat#4311971 Applied Biosystems), mixed, then centrifuged 1 min at RT at 1600 g. The qPCR run was performed on a BioRad CFX384 C1000 Touch Thermocycler (BioRad Hercules, CA) using the following program: 95 °C 10 min, [95 °C 15 s, 60 °C 60 s, 40x]. Data acquisition was performed on the anneal/ extension step. Primer efficiency was determined by plotting the log of the starting template dilution on the X-axis and the mean Cq of the quadruplicate replicates on the Y-axis. The slope of the best fit line was used in the following equation to calculate efficiency: [Efficiency = -1 + 10(-1/slope)]. Primers were accepted if efficiency was 90–105 % and re-designed if efficiency fell outside this range.

For measurement of relative transcript levels, cDNA templates were diluted 1:4 with nuclease-free water then pipetted into quadruplicate wells of a 384 well optical plate for each primer set, hPDGF and the 28 s rRNA reference gene. The 28S rRNA primer pair were Lc-28SF (5'-ACCACTGTTCACACGAAACCCTTC-3') and Lc-28SR (5'-ATCTCGGTTGGATTTTAAACTTTGAAA-3'). The qPCR protocol was performed as above. Analysis of delta delta Cq was performed using BioRad CFX Manager. Mean Cq value was found for each set of 4 replicate wells. The reference gene was utilized to calculate ΔCq. The EF-PDGF control was chosen as the calibrator sample and set to 1. The bar graph represents ΔΔCq (relative normalized expression), with error bars representing standard error of the mean for the replicate values.

Protein analysis

With the PD1 line (Lchsp24-pdgfb), prior to hemolymph collection, adult flies were heat shocked at 37 °C for 2 h with access to water and allowed to recover at room temperature for 3 h. Flies were anesthetized by exposure to carbon dioxide and then one wing was removed. The hemolymph was squeezed into a capillary tube pre-filled with a small amount of cold hemolymph collection buffer (10 mM EDTA pH7 in 1X phosphate-buffered saline (PBS) + 1X Protease inhibitor cocktail (Cat# P2714, Sigma-Aldrich, St. Louis, MO) on ice. Samples were centrifuged 2 min at 10,000 g at 4 °C. Supernatant was transferred to a new tube. Multiple adults were pooled for one sample. Samples were stored at -80 °C.

For offspring of the cross between DR4 driver and EF-PDGF effector, third instar larvae were sorted by fluorescence and rinsed with water on a vacuum funnel with gentle suction. Larvae were briefly placed on a Kim Wipe to remove excess moisture before being placed in Eppendorf tubes and snap frozen in liquid nitrogen. For ES collection, larvae were processed as above and then placed into wells of a 12-well flat-bottomed polystyrene tissue culture plate. Six or 50 larvae were added per well. Two hundred μL (for 6 larvae) or 1 mL (for 50 larvae) of ES collection buffer (Ringer Solution [0.120 mM NaCl, 1.5 mM CaCl 2 , 5 mM KCl, pH 7.4, filter sterilized] + 1X Protease inhibitor cocktail) was added per well, and wells were sealed with an adhesive plate sealer. After 40 min, ES was removed and centrifuged at 21,000 g for 30 min at 4 °C. Supernatant was transferred to a new tube and stored at -80 °C, and debris pellet was discarded. Frozen larvae were lysed on ice in cold gentle lysis buffer [73] using 1 mL glass homogenizers. Following homogenization, lysates were centrifuged at 15,000 g 15 min at 4 °C. Supernatant was transferred to a new tube and stored at -80 °C, while debris pellet was discarded.

Total protein concentration was determined for lysates and hemolymph using the Pierce BCA Protein Assay Kit (Cat#23227, Thermo Fisher, Rockford, IL) and for ES using Quickstart Bradford 1X Dye Reagent and Quickstart Bovine Gamma Globulin Standard (Cat# 500-0205 and 500-0208, BioRad, Hercules, CA). The hPDGF-BB Quantikine ELISA kit was purchased from R&D Systems (Cat# DBB00) and assay was performed according to kit protocol. Lysate and ES samples were assayed in triplicate wells, containing 150 μg and 15–60 μg protein per well respectively. Eighty μg of total protein from each hemolymph sample was assayed per well. Recombinant His-tagged human PDGF-BB (Cat# ab73231, Abcam, Cambridge, MA) was assayed on each ELISA run and provided a positive, in-range control for the integrity of the kit. Sample concentrations were extrapolated or interpolated from a standard curve constructed from the log PDGF-BB concentration (pg/L) on the X-axis and the log mean optical density reading (OD) on the Y-axis. Error bars represent standard error of the mean for the replicate values.

Ethics approval and consent to participate

“Not applicable” as this study did not involve any animal or human data or tissue.

Consent for publication

“Not applicable”.

Availability of data and material

The GenBank accession numbers for the plasmids made in this study are: