All experiments involving animals were conducted under the protocol (SU-ACUC-08032) approved by the Animal Care and Use Committee of Shihezi University. All sheep involved in this research were raised and breed followed the guideline of Animal Husbandry Department of Xinjiang, P.R.China.

shRNAs targeting sheep MSTN were reported in our previous reports [13]. The shMSTN3 (5′-CAAAGATGCTATAAGACAA-3′) targeted the first exon of sheep MSTN gene. The shMSTN3 expression cassette was amplified using primers U6-F (5′-AGT AGT TGC CAG GAT CAC CGT GC-3′) and U6-R (5′-CCT AAT GAG TG A GCT AAC TCA CA-3′), and then cloned into BamH I and SwaI site of ploxP vector to generate ploxP-shMSTN3 (Figure 1).

China Merino sheep fibroblast cells (SF) were isolated and cultured as previously described [14]. 2×10⁵ cells per well were seeded in 12-well plate and cultured in fresh DMEM without antibiotics to achieve 80–90% confluency on the day of transfection. The cells were then transfected with 1.8 µg/well of ploxP-shMSTN3 vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 48 h transfection, cells were split into 100 mm dish at an appropriate dilution for G418 selection (500 µg/ml). Single G418-resistant colonies were obtained after 14 days of selection.

Transgenic fibroblast colonies (TF-s2 and TF-s19) were used to construct transgenic sheep. Sheep nuclear transfer (NT) was performed as described reports [15], [16]. Briefly, ovaries were collected from a local abattoir and transported to our laboratory within 4 h after slaughter. Cumulus-oocyte complexes (COCs) were aspirated from 2 to 5 mm follicles with PBS (containing 5% FCS) by using a 5 ml syringe fitted with a 20-gauge needle. The COCs were cultured in maturation medium at 38.5°C in a humidified atmosphere for 22 h. Cumulus cells were removed by exposure to 1 mg/mL hyaluronidase. Oocytes with a first polar body were enucleated manually in the presence of 7.5 µg/ml of cytochalasin B. A single intact donor cell was injected into the perivitelline space and placed adjacent to the recipient cytoplasm. After injection, reconstructed embryos were transferred into an electrical fusion chamber overlaid with Zimmermann’s fusion medium. Cell fusion was induced with two direct current pulses (1.0 kV/cm, 60 µs, 1 s apart). All fused reconstructed embryos were further activated in 5 µM ionomycin for 4 min, followed by exposure to 1.9 mM 6-dimethylaminopurine in synthetic oviduct fluid with amino acids (SOFaa) for 4 h. Following activation, reconstructed embryos were transferred and cultured in SOFaa. 429 embryos at the 2- to 4-cell stages were surgically transferred into 45 synchronized recipient ewes (7–12 embryos per recipient). Pregnancies were monitored by ultrasound scanning using a trans-abdominal linear probe every two weeks until days 90. Normal control sheep were produced by normal sexual reproduction.

Genomic DNA was isolated from ear biopsy of each lamb using TIANamp Genomic DNA kit (Tiangen Biotech, China). Transgene integration was identified by two independent PCR assays. PCR was performed on 20 ng of genomic DNA using specific primers (Figure 1) for Neo (Neo-F: 5′-ATT CGG CTA TGA CTG GGC ACAC-3′; Neo-R: 5′-CCA GAA AAG CGG CCA TTT TCCA-3′) and for shRNA expression cassette (U6-F: 5′-AGT AGT TGC CAG GAT CAC CGT GC-3′; U6-R: 5′-CCT AAT GAG TGA GCT AAC TCACA-3′). PCR reaction consisted of 95°C for 4 min; 30 cycles at 95°C for 35 s, 58°C for 30 s and 72°C for 40 s; an extension at 72°C for 10 min. PCR products were analyzed by gel electrophoresis.

Biceps brachii muscle tissues were obtained by surgical biopsy from transgenic and control sheep. shRNA expression was identified by RNase protection assay. Small RNAs were isolated from muscle tissues by using the mirVana miRNA isolation Kit (Ambion). The ³²P-labeled RNA probes (29 nt) were generated by in vitro transcription using the mirVana miRNA Probe Construction Kit (Ambion). A DNA oligonucleotide under T7 promoter control, which was reverse complement with the target RNA, was used for in vitro transcription. Protection assay carried out using the mirVana miRNA Detection Kit (Ambion) according to kit manufacturer's instructions. The protected RNA probe was detected by autoradiography.

Protein extracts were taken from transgenic and control biceps brachii muscles, and then were analyzed by western blot analysis as previously described [13]. A primary rabbit anti-MSTN antibody (1∶1000 dilution) (Sigma-Aldrich) and anti-GAPDH (Sigma-Aldrich) were used in the western blotting. Band intensities were estimated by densitometry and corrected by the respective GAPDH band intensities.

Total RNAs were isolated from biceps brachii muscle tissues using Trizol (Invitrogen) according to the manufacturer’s instructions. The primer sets were used for amplifying MHCII (MCF: 5′-AAC GAT ACC GTG GTT GGG-3′; MCR: 5′-CAG CAC GCC GTT ACA CCT-3′), MyoD (MDF: 5′-GCG GAT GAC TTC TAT GAT GACC-3′; MDR: 5′-GTG CAG CGT TTG AGC GTCT-3′), Myogenin (MF: 5′-AAG CGG AAG TCG GTG TCTG-3′; MR: 5′-ATT GTG GGC ATC TGT AGG GT-3′) and Smad2 (sMF: 5′-GGG ATG GAA GAA GTC AGC-3′; sMR: 5′-ATG GGA CAC CTG AAG ACG-3′). Real-Time PCR (Stratagene MX3000P) was carried out using SYBR Green (TaKaRa Biotech, Dalian) following the manufacturer’s protocol. The PCR thermal cycle reactions consisted of denaturation at 95°C for 4 min followed by 45 cycles at 95°C for 15 s, 60°C for 1 min. Cycle threshold (Ct) values were normalized to GAPDH, and comparative quantification of mRNA was done by the ΔΔCt method.

Control sheep (n = 3, male) and three transgenic sheep (male) were weighted at 1, 20, 40, 60 and 90 days after birth. The transgenic M17 showed a fastest increase in body weight and was used for further muscle histological analysis. Biceps brachii muscles were isolated by surgical biopsy from transgenic M17 and control sheep (n = 3). The muscle tissues were fixed in formol for 10 h followed by routine paraffin sectioning and Haematoxilin/Eosin staining. Myofiber diameter and number were determined as previously described [17]. Five representative images (one central and four peripheral) were captured from muscle sections, compounding to 0.7 mm². Myofiber diameter and number were measured with Scion Image software at three independent pictures. Fiber size-distribution diagram was generated and showed as percentage of 500 fibers analyzed. Mean diameter of sheep myofibers represented average value of 500 fibers.

Expression levels of MHCII, MyoD, Myogenin and Smad2 were analysed statistically by one-way ANOVA and Tukey's test. Data were representative of three independent experiments performed in triplicate. Differences were considered significant at P<0.05.

The positive cells were selected as donor cells for constructing cloned embryos by SCNT. The cleavage and blastocyst development rates were 74.4% (131/176) and 14.2% (17/120), respectively. In total, 429 embryos at the 2- to 4-cell stages were transferred to the oviducts of 45 recipient sheep (Table 1). Pregnancy rates were 24.4% (11/45) at forty-five days after embryo transfer. Six recipients were spontaneously aborted and five recipients developed to term. Five lambs were named M17, M18, M21, M23 and M24, respectively. M21 and M24 died 1 h and 22 days after birth, respectively. M17, M18 and M23 have survived for more than six months.

Genomic DNAs were prepared from the ear tissues of transgenic sheep M17, M18 and M23. PCR was used to detect the presence of the transgene. All the three cloned sheep were positive for the shRNA expression cassette and for Neo gene, respectively (Figure 2A, 2B).

To determine whether shRNAs targeting MSTN were expressed in muscles of transgenic sheep, shRNA expression was analyzed by RNase protection assay. As shown in the Figure 2C, shRNA expression was detected in the muscles of three transgenic sheep, whereas not in the muscles of control sheep. shRNA expression was also observed in transgenic fibroblast cells TF-s2 and TF-s19 as positive controls.

To further confirm whether the shRNA inhibited MSTN expression in vivo, biceps brachii muscles were obtained by surgical biopsy from M17, M18, M23 and three control sheep, and MSTN precursor expression was detected by western blot analysis (Figure 2D). Compared with average expression levels of the controls, MSTN expression was reduced by about 61.12%, 31.32% and 10.11% in the transgenic M17, M18 and M23, respectively. This results suggested that MSTN expression was silenced by shRNA in transgenic sheep.

We further determined whether shRNA-mediated MSTN knockdown affected expression of MSTN-related genes in transgenic sheep. Expression levels of MHCII, MyoD, Myogenin and Smad2 were analysed by Real-time RT-PCR. As indicated in Figure 3, expression levels of MHCII, MyoD and Myogenin in transgenic M17 and M18 were significantly higher than that of the controls. Expression levels of Smad2 were decreased in transgenic sheep, but without significant difference compared with the controls.

Based on body weight records, transgenic sheep showed a faster increase tendency in body weight than control sheep during the 90 days after birth (Figure 4). The 3-month-old M17 (25.42 kg), M18 (23.52 kg) and M23 (22.93 kg) were heavier than control sheep (21.22 kg, n = 3), respectively. Daily weight gain of M17 was 0.23 kg/d, when compared with 0.18 kg/d of control sheep.

Muscle tissues of M17 were isolated by surgical biopsy and sectioned for morphometric analysis. H&E staining showed larger myofiber sizes in the transgenic sheep (Figure 5A). Digital morphometric analysis of 500 myofibers from M17 muscle tissues revealed an increase in myofiber diameter compared with control sheep (Figure 5B). More myofibers were in the large myofiber diameter range (35–65 µm) compared with controls (Figure 5B). Mean diameter of M17 myofibers was 58 µm, whereas that of control myofibers was 47 µm (Figure 5C). No significant difference was found in the myofiber number per an unbiased 0.7 mm² frame between M17 (912±13) and controls (869±16; n = 3). These results suggested that MSTN suppression may caused hypertrophy in transgenic sheep.