2.1 Construction of duck SOST recombinant adenovirus vector
After predicting the restriction enzyme sites (http://nc2.neb.com/NEBcutter2/) of duck SOST coding domain sequence (CDS, XM_005026106), the NEBuilder (http://nebuilder.neb.com/) was used to design primers with overlapping sequences of pAdTrack-CMV at both ends (Table S1). The entire CDS of duck SOST was amplified by PCR. After sequencing, the amplified PCR products of SOST and pAdTrack-CMV plasmid, linearized with XhoI and KpnI (Takara, China), were ligated E. coli DH5a homologous recombinase (Biodragon Immunotechnologies Co., Ltd, China). Then, the recombinant plasmids were confirmed by sequencing and digestion. The recombinant plasmids were named pAdTrack-CMV-duSOST. The pAdTrack-CMV-duSOST linearized with PmeI and the adenoviral backbone plasmid (pAdEasy-1) were ligated with homologous recombinant in E. coli BJ5183 (Miaoling Bioscience & Technology Co., Ltd, China). The second recombinant plasmid was identified by PacI (NEB, China) restriction enzyme digestion and sequenced, which showed that the vector was successfully constructed and named pAd-duSOST (Fig. 1a). Finally, the second recombinant plasmid was subsequently used to generate recombinant adenoviruses in human embryonic kidney cells (HEK293; Kunming Institute of Zoology, China). The specific steps of adenovirus packaging in HEK293 cells are provided in the supplementary files.
2.2 The titer determination of recombinant adenovirus
The titer of adenovirus was measured according to Luo's article . The HEK-293 cells were inoculated at 1×104 cells/well in a 96-well plate and 90 µL/well complete culture medium containing 10% FBS after cell culture for 24 hours. Then, 10 µL virus was added into the first well, and ten-fold dilution of the previous virus-containing solution was performed before the virus was added into the next well (12 wells in each group, altogether 3 groups)༎After 48 hours, an inverted fluorescence microscope (OLYMPUS IX73, Japan) was used to observe the fluorescence-positive cells in each well༎The virus titer was calculated according to the following equation; virus titer (U/mL) = m×10n + 1, where n is the serial number of the reference well, and m is the number of positive cells༎
2.3 Duck myoblast isolation and culture
The fertilizer eggs of ducks were collected from the Waterfowl Breeding Farm of Sichuan Agricultural University. The eggs were kept for incubating for 13 days under the same hatching conditions. According to the procedures of our previous studies, the isolation and cell culture of duck myoblasts were according to the guidelines of our previous studies . The primary myoblasts of ducks were cultured in a complete growth medium containing DMEM/F-12 (HyClone, China), 10% FBS, and 1% penicillin/streptomycin, and then were incubated in an incubator with 5% CO2 at 37°C. All animal experiments were allowed by the Animal Ethics Committee of Sichuan Agricultural University.
2.4 Multiplicity of infection (MOI) determination
The duck primary myoblasts were inoculated at 1×105 cells/well in a 6-well plate, growing myoblasts (70%-80% confluent) were infected with adenovirus solution of 30, 40, 50, 60, and 70 multiplicity of infection (MOI), respectively. The cells were collected after 48 hours of infection, and the mRNA expression of the SOST gene in the cells was measured by real-time PCR.
2.5 Transfection of pAd-duSOST to myoblasts
The duck primary myoblasts were inoculated at 1×105 cells/well in a 6-well plate and cultured in a complete medium (DMEM/F12 + 10% FBS + 100U/mL penicillin and 100 g/mL streptomycin). They were divided into the Ad-duSOST treatment group and the Ad-NC control group, with three replicates in each group. For proliferation assay, growing myoblasts (50% confluent) were infected with optimal MOI of Ad-duSOST and Ad-NC. At 24 hours post-infected, cells were harvested for sampling. For differentiation assay, the culture medium was changed to differentiation medium (DM, DMEM/F12 containing 2% horse serum and 1% penicillin/ streptomycin) until approximately 80% confluence was reached, and the optimal MOI of Ad-duSOST and Ad-NC virus solution was added to each group. All cells were cultured in the incubator with 5% CO2 at 37°C. The cell samples infected by the virus were collected and stored at -80 ℃ for RNA isolation.
2.6 Cell proliferation assay
Myoblasts viability was analyzed by a Cell Counting Kit-8 (CCK8, Bestbio Biotechnology, China). The primary myoblasts were seeded into a 96-well plate with a density of 5×103 cells/well; after transfection Ad-duSOST or Ad-NC, myoblasts were incubated with 10 µL CCK-8 for additional 3 hours at 37°C; the absorbance at 450 nm was measured using a Microplate Reader (Thermo, USA). The samples from each treatment had six replicates at each time point.
Duck myoblasts were inoculated into 96-well plates with 2000–3000 cells per well with three repeats in each group. After 24 hours of treatment, the cells were incubated with EdU medium for 3 hours and then fixed with 4% paraformaldehyde. After that, the test was carried out according to the instructions of the EdU kit (Ribobio, China). Finally, it was observed and photographed under a fluorescence microscope.
2.7 Flow cytometry assay
Total 1×105 myoblasts were inoculated in a 6-well plate and transfected when the cell density was about 40–50%. After 24 hours of transfection, the cells were digested with trypsin and collected. Then, the cells were washed once with PBS, centrifuged at 300 rpm for 5 min, and then the supernatant was discarded. The precipitation was fixed with ice-precooled 70% ethanol at 4℃ for 18 hours. The samples were centrifuged at 300 rpm for 5 min, and the precipitate at the bottom was suspended with 1 mL PBS and filtered with a 300-mesh sieve. After centrifugation for 5 min at 300 rpm, PBS was discarded, and the precipitate was stained with 0.5 mL PI solution for 15 min (37℃, avoid light). The number of cells in each stage was analyzed by flow cytometry at 488 nm laser wavelength.
Myoblasts were switched to differentiation medium (DM) for 2 days infected with Ad-duSOST or Ad-NC, and then these cells were used for immunofluorescence (IF) staining. The cells were fixed with 4% paraformaldehyde (PFA) for 30 min and treated with 0.1% Triton X-100 for 5 min at room temperature, then blocked with 5% BSA for 1 hour. After incubation overnight at 4℃ in a mouse primary polyclonal antibodies against MYHC (diluted 1:500; Biosynthesis Biotechnology, China), the cells were incubated against Cy3-conjugated secondary antibody (diluted 1:500; Beyotime Biotechnology, China). The slides were co-stained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma) to visualize the nuclei. Then, the cells were visualized with an immunofluorescence microscope after having been washed in PBS thrice. The relative area of positive staining was evaluated with Image-Pro Plus 6.0 software (Media Cybernetics Corporation, USA).
2.9 Western blot
Total proteins were isolated by lysing cells in ice-cold RIPA lysis buffer. Protein samples from individual experiments were pooled for western blotting analysis. The primary antibodies were used; anti-MYHC (1:500 dilutions) and anti-GAPDH (1:1000 dilutions). The membranes were incubated with antibodies at 4°C overnight and washed in washing buffer (10 mM Tris-HCI, pH 7.5, 100 mM NaCl, and 0.1% Tween 20). Next, the membranes were treated with horseradish peroxidase-conjugated IgG antibody (1:2000 dilutions) for 2 hours at room temperature. Each experiment was biologically replicated for triplicate times. Densitometry analysis of the bands relative to GAPDH was performed using Image J software (National Health Institute, Bethesda, MD, USA).
2.10 Real-time PCR
Total RNA was extracted from myoblasts by RNAiso plus (Takara, Japan) according to the manufacturer's instructions and then measured by spectrophotometry. RNA was reverse-transcribed to synthesize the cDNA using the reverse transcript system (Takara, China). Real-time PCR was carried out with the SYBR Prime Script RT-PCR Kit (Takara, China) using the Bio-Rad CFX Manager (Bio-Rad Laboratories, USA). Expression of duck SOST, CDK6, PCNA, and CCND1/2 were detected, β-actin and GAPDH were used as inner controls. One sample collected from cells was repeated in triplicate. Primer sequences for real-time PCR are provided in Table S3. The relative expression levels of each gene were analyzed by the 2−ΔΔCt method.
Two days' treatment after the myoblasts transfected with Ad-duSOST and Ad-NC, the cells were collected for sampling. Each treatment has three replicate samples. RNA was isolated, and its concentration and integrity were measured. High-quality RNA was sent to Novogene (Beijing, China) for cDNA libraries construction and sequencing. RNA-seq libraries were then generated. Briefly, after RNA fragmentation, double-stranded cDNA was synthesized by replacing dTTPs (deoxythymidine triphosphate) with dUTPs (deoxyuridine triphosphate) in reaction buffer used for second-strand cDNA synthesis. The resulting double-stranded cDNA was ligated to adaptors after being end-repaired and A-tailed. Single-strand cDNA was then obtained using the USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, U.K.). Finally, PCR amplification was performed to enrich the cDNA libraries. Sequencing was performed on an Illumina Hiseq 2500 instrument to generate 150-bp paired-end reads.
2.12 Mapping and differentially expressed genes (DEGs) analysis
Hisat2 (v2.1.0) built the index of genome files and mapped RNA-seq data to the reference genome (IASCAAS_PekingDuck_PBH1.5, GCF_003850225.1). StringTie (v1.3.3b) assembles the alignments into full and partial transcripts, and estimates the expression levels of all genes and transcripts, and creates read in files for Ballgown. Finally, the software packages in R (v3.5.1), such as gene filter, dplyr, and devtools, were used to analyze the differential genes. Functional groups and pathways encompassing the DEGs were identified based on GeneOntology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using the KOBAS 3.0 software. The threshold was set as a P ≤ 0.05.
2.13 Statistical analysis
The data were subjected to analysis of variance (ANOVA), and the means were compared for significance by Tukey's test using SPSS22 (IBM Corp, Armonk, NY). The results were expressed as the mean ± SEM. A p-value of less than 0.05 was considered statistically significant.