CircRNA-5335 Regulates the Differentiation and Proliferation of Sheep Preadipocyte via the miR-125a-3p/STAT3 Pathway.

The content of intramuscular fat (IMF) from preadipocytes is proportional to meat quality in livestock. However, the roles of circRNAs in IMF deposition in sheep are not well known. In this study, we show that circRNA-5335/miR-125a-3p/STAT3 play a crucial adjective role in the proliferation and differentiation of sheep preadipocytes. In this study, we characterized the roles of differentially expressed circRNA-5335/miR-125a-3p/STAT3, which were screened from sheep of different months of age and based on sequencing data. Firstly, the expression profiles of circRNA-5335/miR-125a-3p/STAT3 were identified during the differentiation of preadipocytes in vitro by RT-qPCR and WB. Then, the targeting relationship of the circRNA-5335/miR-125a-3p/STAT3 was verified by dual-luciferase reporter assays. The results of RT-qPCR, CCK8, EdU and Oil Red O staining assay showed that miR-125a-3p suppressed the differentiation and raised the proliferation of preadipocytes by targeting STAT3. As a competing endogenous RNA, the downregulation of circRNA-5335 decreased the expression of STAT3 by increasing miR-125a-3p, which inhibited the differentiation of preadipocytes and promoted proliferation. Our present study demonstrates the functional significance of circRNA-5335/miR-125a-3p/STAT3 in the differentiation of sheep preadipocytes, and provides novel insights into exploring the mechanism of IMF.

DLL4/Notch blockade disrupts mandibular advancement-induced condylar osteogenesis by inhibiting H-type angiogenesis.

BACKGROUND: Blocking Delta-like 4 (DLL4)/Notch has emerged as a promising therapeutic target for the treatment of tumours by deregulating angiogenesis. However, DLL4/Notch serves as a negative regulator of angiogenesis in multiple organs while acting as a positive regulator of H-type angiogenesis in postnatal long bones. Therefore, the effect of DLL4/Notch signalling blockade on mandibular condylar osteogenesis attracted our attention. OBJECTIVE: To explore the effect of blocking DLL4/Notch on mandibular advancement (MA)-induced condylar osteogenesis. METHODS: Six-week-old young male C57BL/6J mice (n = 40) were randomly divided into four groups: control group, MA group, MA + Anti-DLL4 group and MA + IgG group. Of note, IgG served as the isotype control for the anti-DLL4. The femurs, tibias and mandibular condyles were collected after sacrificing mice on Day 31 for morphology, micro-computed tomography, immunofluorescence, histology and immunohistochemistry evaluation. RESULTS: First, DLL4/Notch blockade shortened femoral length and reduced bone mass by inhibiting H-type angiogenesis. Second, DLL4/Notch blockade disrupted MA-induced condylar head volume and quality by inhibiting H-type angiogenesis. Mechanistically, blocking DLL4/Notch reduced the number of runt-related transcription factor 2+ (RUNX2+ ) early osteoprogenitors and the expression of Noggin protein in the condylar subchondral bone by inhibiting H-type angiogenesis. In addition, blockade of DLL4/Notch also destroyed the condylar cartilage layer. CONCLUSION: DLL4/Notch blockade results in shortened femurs and osteopenia, as well as impaired MA-induced condylar osteogenic volume and quality in growing mice by inhibiting H-type angiogenesis. Therefore, when blocking DLL4/Notch is used as a treatment target for diseases, attention should be paid to its impact on the bone mass of mandibular condyle.

Identification and analysis of differentially expressed microRNAs in endometrium to explore the regulation of sheep fecundity.

BACKGROUND: MicroRNAs (miRNAs) play an important regulatory role in mammalian reproduction. Currently, most studies are primarily concentrated on ovarian miRNAs, ignoring the influence of endometrial miRNAs on the fecundity of female sheep. To uncover potential regulators of sheep fecundity, RNA-seq was used to comparatively analyze miRNA expression profiles of endometrium between high prolificacy sheep (HP, litter size = 3) and low prolificacy sheep (LP, litter size = 1) with FecB genotype. RESULTS: Firstly, genomic features of miRNAs from endometrium were analyzed. Furthermore, 58 differentially expressed (DE) miRNAs were found in the endometrium of Hu sheep with different litter size. A co-expression network of DE miRNAs and target genes has been constructed, and hub genes related litter size are included, such as DE miRNA unconservative_NC_019472.2_1229533 and unconservative_NC_019481.2_1637827 target to estrogen receptor α (ESR1) and unconservative_NC_019481.2_1637827 targets to transcription factor 7 (TCF7). Moreover, functional annotation analysis showed that the target genes (NRCAM and NEGR1) of the DE miRNAs were significantly enriched in cell adhesion molecules (CAMs) signaling pathway, which was related to uterine receptivity. CONCLUSION: Taken together, this study provides a new valuable resource for understanding the molecular mechanisms underlying Hu sheep prolificacy.

Comparison of part-time and full-time mandibular advancement: enlightenment based on type H vessel coupling osteogenesis.

OBJECTIVES: This study was aimed at resolving the controversy over the wearing time of mandibular advancement (MA) appliances by comparing the differences between part-time and full-time MA (PTMA and FTMA) from the perspective of promoting H-type vessel coupling osteogenesis in the condylar heads. MATERIALS AND METHODS: Thirty 30-week-old male C57BL/6J mice were randomly divided into 3 groups: the control (Ctrl), PTMA, and FTMA groups. The mandibular condyles were analyzed by morphology, micro-computed tomography, histological staining, and immunofluorescence staining to investigate the changes of the condylar heads in the PTMA and FTMA groups after 31 days. RESULTS: Both PTMA and FTMA models promoted condylar growth and achieved stable mandibular advancement at day 31. However, compared with PTMA, FTMA has the following characteristics. First, new bone formation in the condylar head was detected in the retrocentral region in addition to the posterior region. Second, the condylar proliferative layer was thicker, and the number of pyknotic cells in the hypertrophic and erosive layers was higher. Moreover, endochondral osteogenesis of the condylar head was more active. Finally, the retrocentral and posterior regions of the condylar head had more vascular loops or arcuate H-type vessel coupling Osterix+ osteoprogenitors. CONCLUSIONS: While both PTMA and FTMA induced new bone formation in the condylar heads of middle-aged mice, FTMA promoted more osteogenesis by volume and region. Furthermore, FTMA presented more H-type vessel coupling Osterix+ osteoprogenitors in both the retrocentral and posterior regions of the condylar head. CLINICAL RELEVANCE: FTMA is better at promoting condylar osteogenesis, especially in non-growing patients. We suggest that enhancing H-type angiogenesis could be an effective strategy to achieve favorable MA outcomes, especially for patients unable to meet the FT-wearing requirement or being non-growing.

Deferoxamine mesylate enhances mandibular advancement-induced condylar osteogenesis by promoting H-type angiogenesis.

BACKGROUND: The effect of functional orthopaedic treatment for mandibular deficiency relies on mandibular advancement (MA)-induced condylar new bone formation. However, this is not easy to achieve, especially in non-growing patients. Therefore, how to obtain reliable MA-induced condylar osteogenesis is a subject much worthy of study. OBJECTIVE: To investigate whether deferoxamine mesylate (DFM) enhances MA-induced condylar osteogenesis in middle-aged mice. METHODS: Forty 30-week-old male C57BL/6J mice were randomly divided into 4 groups: the control (Ctrl), DFM, MA + Ctrl and MA + DFM groups. After a 4-week experimental period, femurs, tibias and condyles were collected for morphological, micro-computed tomography and histological evaluation. RESULTS: For long bones, DFM reversed osteoporosis in middle-aged mice by promoting H-type angiogenesis. For mandibular condyles, MA promoted condylar osteogenesis in middle-aged mice, thereby allowing the mandible to achieve a stable protruding position. In addition, DFM enhanced the volume and quality of MA-induced condylar new bone formation. Furthermore, histological analysis revealed that DFM enhanced MA-induced condylar subchondral ossification. Mechanistically, it was confirmed that DFM increased the number of H-type vessels and their coupled Osterix+ osteoprogenitors by upregulating the hypoxia-inducible factor (HIF)-1α signalling pathway, thereby enhancing MA-induced condylar osteogenesis. CONCLUSION: Applying DFM to enhance MA-induced condylar osteogenesis through H-type angiogenesis is expected to be an effective strategy to achieve favourable functional orthopaedic treatment effectiveness in non-growing patients.

Comparative Analysis of the Liver Transcriptome of Beijing You Chickens and Guang Ming Broilers under Salmonella enterica Serovar Typhimurium Infection.

Salmonella enterica serovar Typhimurium (ST) is a food-borne pathogen that can infect animals and humans. It is currently the most common bacterial pathogen that negatively affects the poultry industry. Although different chicken breeds have been observed to exhibit diverse resistance to ST infection, the underlying genetic mechanisms remain unclear and the genes involved in this differential disease resistance need to be identified. To overcome this knowledge gap, we used a liver transcriptome analysis to screen differentially expressed genes (DEGs) in two different chicken breeds (local Beijing You (BY) and commercial Guang Ming No. 2 broiler line B (GM)) before and after ST infection. We also performed weighted gene co-expression network analysis (WGCNA) to detect hub genes, and employed selection signal analysis of candidate genes. Three promising genes (EGR1, JUN and FOS) were eventually identified, and were significantly and differentially expressed in the same breed under different conditions, and in the two breeds after ST infection. Hub genes, such as PPFIA4 and ZNF395, were identified using WGCNA, and were associated with the ratio of heterophils to lymphocytes (H/L), an indicator of disease resistance. the present study identified several genes and pathways associated with resistance to ST infection, and found that BY had greater resistance to ST infection than GM. The results obtained provide valuable resources for investigating the mechanisms of resistance to ST infection in different chicken breeds.

Biological mechanism of surgery-mediated acceleration of orthodontic tooth movement: A narrative review.

Surgery-mediated acceleration of orthodontic tooth movement (SAOTM) has been proven effective for decades. Research has confirmed that surgical approaches play an important role in adult patients with a short orthodontic treatment time. The mechanism of SAOTM involves short-term acceleration of localized hard and soft tissue remodeling, known as the regional acceleratory phenomenon. However, no relevant review on the biological mechanism of SAOTM has been performed to date. The proposed biological mechanism of acceleration of OTM involves the participation of various cells, cytokines, and signaling pathways. We herein review the relevant literature and summarize the biological mechanism of SAOTM to provide new insights for further research on acceleration of OTM.

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System.

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Transcriptomic Analysis Reveals the Involvement of lncRNA-miRNA-mRNA Networks in Hair Follicle Induction in Aohan Fine Wool Sheep Skin.

Long non-coding RNAs (lncRNA) and microRNAs (miRNA) are new found classes of non-coding RNAs (ncRNAs) that are not translated into proteins but regulate various cellular and biological processes. In this study, we conducted a transcriptomic analysis of ncRNA and mRNA expression in Aohan fine wool sheep (AFWS) at different growth stages (embryonic day 90, embryonic day 120, and the day of birth), and explored their relationship with wool follicle growth. In total, 461 lncRNAs, 106 miRNAs, and 1,009 mRNAs were found to be differentially expressed during the three stages of wool follicle development. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to clarify the roles of the differentially expressed lncRNA, miRNA, and mRNA in the different stages of wool follicle development. Quantitative real-time PCR (qRT-PCR) was used to validate the results of RNA-seq analysis. lncRNA (MSTRG.223165) was found to act as a competing endogenous RNA (ceRNA) and may participate in wool follicle development by acting as an miR-21 sponge. Network prediction implicated the MSTRG.223165-miR-21-SOX6 axis in the wool follicle development. The targeting relationships of miR-21 with SOX6 and MSTRG.223165 were validated in dual-luciferase assays. This is the first report indicating the association of the lncRNA-miRNA-mRNA network with wool follicle development in AFWS. This study provides new insights into the regulation of the wool follicle growth and represents a solid foundation for wool sheep breeding programs.

Effects of the Forsus Fatigue-resistant Device on Skeletal Class II Malocclusion Correction.

AIM: To test the hypothesis that skeletal and dentoalveolar effects are both important in skeletal class II malocclusion corrected with the Forsus fatigue-resistant device (FRD). MATERIALS AND METHODS: A total of 35 patients (16 females and 19 males; age 12.0 ± 0.6 years) with skeletal class II malocclusion treated with the Forsus FRD were included. Lateral cephalometric radiographies before and after treatment were collected. Cephalometric analysis and superimpositions were applied. Pancherz's analysis was performed to discover the skeletal and dentoalveolar effects on all patients and 60% contribution was set as a milestone to classify. Statistical comparisons were performed by paired t testing (p < 0.05). RESULTS: The mean treatment period of the Forsus FRD was 6.4 ± 0.2 months. All patients (AG) have been corrected to class I molar relationship in three mechanisms: 15 patients in the skeletal group (SG), 10 patients in the dentoalveolar group (DG), and 10 patients in the skeletal and dentoalveolar group (SDG). Four groups showed a significant change in skeletal sagittal relationship improvement (p < 0.05). The AG, SG, and SDG showed a significant improvement in the growth of the mandible (Co-Go, Go-Pog, and Co-Gn, p < 0.05). The DG showed a significant improvement in the growth of the mandibular body (Go-Pog, p < 0.05). CONCLUSION: Three mechanisms were found in skeletal class II malocclusion corrected with the Forsus FRD. Skeletal and dentoalveolar effects are both important in skeletal class II malocclusion corrected with the Forsus FRD. And skeletal and dentoalveolar effects played differential roles in different cases. CLINICAL SIGNIFICANCE: The mechanism of skeletal class II correction with Forsus FRD may divide into mandibular growth, dentoalveolar effects, and both.

Discovery of genes and proteins possibly regulating mean wool fibre diameter using cDNA microarray and proteomic approaches.

Wool fibre diameter (WFD) is one of the wool traits with higher economic impact. However, the main genes specifically regulating WFD remain unidentified. In this current work we have used Agilent Sheep Gene Expression Microarray and proteomic technology to investigate the gene expression patterns of body side skin, bearing more wool, in Aohan fine wool sheep, a Chinese indigenous breed, and compared them with that of small tail Han sheep, a sheep bread with coarse wool. Microarray analyses showed that most of the genes likely determining wool diameter could be classified into a few categories, including immune response, regulation of receptor binding and growth factor activity. Certain gene families might play a role in hair growth regulation. These include growth factors, immune cytokines, solute carrier families, cellular respiration and glucose transport amongst others. Proteomic analyses also identified scores of differentially expressed proteins.

Identification and characterization of circRNAs in the skin during wool follicle development in Aohan fine wool sheep.

BACKGROUND: Aohan fine wool sheep (AFWS) is a historically bred fine wool sheep, cultivated in China. The wool has excellent quality and good textile performance. Investigating the molecular mechanisms that regulate wool growth is important to improve wool quality and yield. Circular RNAs (circRNAs) are widely expressed non-coding RNAs that can act as competitive endogenous RNAs (ceRNAs) to bind to miRNAs. Although circRNAs have been studied in many fields, research on their activity in sheep wool follicles is limited. To understand the regulation of circRNAs in the growth of fine wool in sheep, we used RNA-Seq to identify circRNAs in sheep shoulder skin samples at three developmental stages: embryonic day 90 (E90d), embryonic day 120 (E120d), and at birth (Birth). RESULTS: We identified 8753 circRNAs and found that 918 were differentially-expressed. We then analyzed the classification and characteristic of the circRNAs in sheep shoulder skin. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we identified the source genes of circRNAs, which were mainly enriched in cellular component organization, regulation of primary metabolic processes, tight junctions, and the cGMP-PKG and AMPK signaling pathways. In addition, we predicted interactions between 17 circRNAs and eight miRNAs, using miRanda software. Based on the significant pathways, we speculate that circ_0005720, circ_0001754, circ_0008036, circ_0004032, circ_0005174, circ_0005519, and circ_0007826 might play an important role in regulating wool follicle growth in AFWS. Seven circRNAs were randomly selected to validate the RNA-Seq results, using qRT-PCR. CONCLUSION: Our results provide more information about circRNAs regulation of wool follicle development in AFWS, and establish a solid foundation for future research.

Effects of chlorogenic acid-enriched extract from Eucommia ulmoides Oliver leaf on growth performance and quality and oxidative status of meat in finishing pigs fed diets containing fresh or oxidized corn oil.

To investigate the effects of chlorogenic acid-enriched extract (CGAE) from Eucommia ulmoides Oliver leaf on growth performance and quality and oxidative status of meat in pigs fed diets containing fresh or oxidized corn oil, a total of 180 barrows (initial body weight: 81.6 ± 2.08 kg) were randomly allocated into 6 diet treatments (5 replicate pens per treatment and 6 barrows per pen) in a 2 × 3 factorial design with corn oil (fresh or oxidized corn oil at 5% inclusion of diet) and CGAE (0, 500 or 1,000 mg/kg of diet containing fresh or oxidized corn oil) as main factors. The experiment lasted for 6 weeks. Dietary oxidized oil reduced average daily gain (ADG, p < .05) and average daily feed intake (ADFI, p < .01) of pigs and pH24 (p < .05), total antioxidant capacity (T-AOC, p < .01), glutathione peroxidase (GPx, p < .05) and sarcoplasmic reticulum Ca2+ -ATPase (SERCA, p < .05) activities in meat and increased drip loss (p < .01), cooking loss (p < .05), malondialdehyde (p < .01) and carbonyl (p < .01) contents and mRNA expression of superoxide dismutase 1 (SOD1, p < .05) in meat. Dietary CGAE supplementation at 1,000 mg/kg increased (p < .05) ADG and ADFI of pigs and pH24 , T-AOC, T-SOD, GPx and SERCA activities and mRNA expression of SOD1 in meat and reduced (p < .05) drip loss, cooking loss, carbonyl and malondialdehyde contents in meat. No interaction effects between oxidized corn oil and CGAE were found in pigs. Overall, dietary CGAE supplementation at 1,000 mg/kg improved growth performance and quality and oxidative status of meat in pigs subjected or not to oxidative stress induced by dietary oxidized oil.

Application of oligonucleotides to construct a conditional targeting vector for porcine IκBα.

Conditional gene targeting at porcine IκBα may be a solution to delayed xenograft rejection, the main barrier to xenotransplantation. An oligonucleotide­based method was applied to construct the vector for conditional targeting of porcine IκBα. This method was free from PCR amplification during the assembling of the different vector elements, avoiding introduction of unwanted mutations. With the help of short double­stranded DNA fragments produced by annealing oligonucleotides, nondirectional cloning has also been avoided. By making the best of directional cloning, a highly complex targeting vector was built within 3 weeks. The present study also explained why the two recombination­based methods (recombineering and gateway recombination), although having demonstrated to be highly efficient in constructing ordinary targeting vectors, were not appropriate in this context. The description in the present study of an additional method to efficiently construct targeting vectors is suggested to introduce more flexibility in the field therefore helping to meet the different needs of the researchers.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Identification of differentially expressed genes affecting hair and cashmere growth in the Laiwu black goat by microarray.

Goats are an important source of fibers. In the present study microarray technology was used to investigate the potential genes primarily involved in hair and cashmere growth in the Laiwu black goat. A total of 655 genes differentially expressed in body (hair­growing) and groin (hairless) skin were identified, and their potential association with hair and cashmere growth was analyzed. The majority of genes associated with hair growth regulation could be assigned to intracellular, intracellular organelle, membrane­bound vesicle, cytoplasmic vesicle, pattern binding, heparin binding, polysaccharide binding, glycosaminoglycan binding and cytoplasmic membrane­bound vesicle categories. Numerous genes upregulated in body compared with groin skin contained common motifs for nuclear factor 1A, Yi, E2 factor (E2F) and cyclic adenosine monophosphate response element binding (CREB)/CREBß binding sites in their promoter region. The promoter region of certain genes downregulated in body compared with groin skin contained three common regions with LF­A1, Yi, E2F, Collier/Olfactory­1/early B­cell factor 1, peroxisome proliferator­activated receptor α or U sites. Thus, the present study identified molecules in the cashmere­bearing skin area of the Laiwu black goat, which may contribute to hair and cashmere traits.

Safety Evaluation of Neo Transgenic Pigs by Studying Changes in Gut Microbiota Using High-Throughput Sequencing Technology.

The neo (neomycin phosphotransferase) gene is widely used as a selection marker in the production of genetically engineered animals and plants. Recent attention has been focused on safety concerns regarding neo transgene expression. In this study, neo transgenic and non-transgenic piglets were randomly assigned into Group A and Group B to evaluate effects of neo transgene by studying changes in gut microbiota using high-throughput sequencing. Group A pigs were fed a standard diet supplemented with antibiotic neomycin; Group B pigs were fed a standard diet. We examined horizontal transfer of exogenous neo gene using multiplex PCR; and investigated if the presence of secreted NPT II (neo expression product) in the intestine could lead to some protection against neomycin in transgenic pigs by monitoring different patterns of changes in gut microbiota in Group A animals. The unintended effects of neo transgene on gut microbiota were studied in Group B animals. Horizontal gene transfer was not detected in gut microbiota of any transgenic pigs. In Group A, a significant difference was observed between transgenic pigs and non-transgenic pigs in pattern of changes in Proteobacteria populations in fecal samples during and post neomycin feeding. In Group B, there were significant differences in the relative abundance of phyla Firmicutes, Bacteroidetes and Proteobacteria, and genera Lactobacillus and Escherichia-Shigella-Hafnia between transgenic pigs and non-transgenic pigs. We speculate that the secretion of NPT II from transgenic tissues/cells into gut microbiota results in the inhibition of neomycin activity and the different patterns of changes in bacterial populations. Furthermore, the neo gene also leads to unintended effects on gut microbiota in transgenic pigs that were fed with basic diet (not supplemented with neomycin). Thus, our data in this study caution that wide use of the neo transgene in genetically engineered animals should be carefully considered and fully assessed.

Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs.

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN(-/-)) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN(+/+)) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs.

Transgenic pigs play an important role in producing higher quality food in agriculture and improving human health when used as animal models for various human diseases in biomedicine. Production of transgenic pigs, however, is a lengthy and inefficient process that hinders research using pig models. Recent applications of the CRISPR/Cas9 system for generating site-specific gene knockout/knockin models, including a knockout pig model, have significantly accelerated the animal model field. However, a knockin pig model containing a site-specific transgene insertion that can be passed on to its offspring remains lacking. Here, we describe for the first time the generation of a site-specific knockin pig model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. We also report a new genomic "safe harbor" locus, named pH11, which enables stable and robust transgene expression. Our results indicate that our CRISPR/Cas9 knockin system allows highly efficient gene insertion at the pH11 locus of up to 54% using drug selection and 6% without drug selection. We successfully inserted a gene fragment larger than 9 kb at the pH11 locus using the CRISPR/Cas9 system. Our data also confirm that the gene inserted into the pH11 locus is highly expressed in cells, embryos and animals.