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Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause significant economic losses to the global swine industry, yet effective prevention and control measures remain elusive. The development of novel antivirals is thus urgently needed. Rifampicin (RFP), a semisynthetic derivative of rifamycin, has been previously reported to inhibit the replication of certain mammalian DNA viruses as well as RNA viruses. In this study, we unveil RFP as a potent inhibitor of PRRSV both in Marc-145 cells (half-maximal inhibitory concentration 61.26 µM) and porcine alveolar macrophages (half-maximal inhibitory concentration 53.09 µM). The inhibitory effect of RFP occurred during viral replication rather than binding, internalization and release. We also demonstrated that RFP inhibits PRRSV proteins production in the early stage of infection, without inhibiting host protein synthesis. Moreover, RFP effectively restricted porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV) infection in Vero cells. In summary, these findings indicate the promising potential of RFP as a therapeutic agent for PRRSV, PEDV and PEAV infection in pig farms.
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Ubiquitin-conjugation enzyme E2C (UBE2C) is a crucial component of the ubiquitin-proteasome system that is involved in numerous cancers. In this study, we find that UBE2C expression is significantly increased in mouse embryos, a critical stage during skeletal muscle development. We further investigate the function of UBE2C in myogenesis. Knockdown of UBE2C inhibits C2C12 cell differentiation and decreases the expressions of MyoG and MyHC, while overexpression of UBE2C promotes C2C12 cell differentiation. Additionally, knockdown of UBE2C, specifically in the tibialis anterior muscle (TA), severely impedes muscle regeneration in vivo. Mechanistically, we show that UBE2C knockdown reduces the level of phosphorylated protein kinase B (p-Akt) and promotes the degradation of Akt. These findings suggest that UBE2C plays a critical role in myoblast differentiation and muscle regeneration and that UBE2C regulates myogenesis through the Akt signaling pathway.
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Diferenciación Celular , Desarrollo de Músculos , Músculo Esquelético , Mioblastos , Proteínas Proto-Oncogénicas c-akt , Regeneración , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras , Animales , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Regeneración/genética , Mioblastos/metabolismo , Mioblastos/citología , Ratones , Desarrollo de Músculos/genética , Línea CelularRESUMEN
Intramuscular fat (IMF) plays a crucial role in enhancing meat quality, enriching meat flavor, and overall improving palatability. In this study, Single-cell RNA sequencing was employed to analyze the longissimus dorsi (LD) obtained from Guangdong small-ear spotted pigs (GDSS, with high IMF) and Yorkshire pigs (YK, with low IMF). GDSS had significantly more Fibro/Adipogenic Progenitor (FAPs), in which the CD9 negative FAPs (FAPCD9-) having adipogenic potential, as demonstrated by in vitro assays using cells originated from mouse muscle. On the other hand, Yorkshire had more fibro-inflammatory progenitors (FIPs, marked with FAPCD9+), presenting higher expression of the FBN1-Integrin α5ß1. FBN1-Integrin α5ß1 could inhibit insulin signaling in FAPCD9-, suppressing adipogenic differentiation. Our results demonstrated that fat-type pigs possess a greater number of FAPCD9-, which are the exclusive cells in muscle capable of differentiating into adipocytes. Moreover, lean-type pigs exhibit higher expression of FBN1-Integrin α5ß1 axis, which inhibits adipocyte differentiation. These results appropriately explain the observed higher IMF content in fat-type pigs.
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Coat colour largely determines the market demand for several cat breeds. The KIT proto-oncogene (KIT) gene is a key gene controlling melanoblast differentiation and melanogenesis. KIT mutations usually cause varied changes in coat colour in mammalian species. In this study, we used a pair of single-guide RNAs (sgRNAs) to delete exon 17 of KIT in somatic cells isolated from two different Chinese Li Hua feline foetuses. Edited cells were used as donor nuclei for somatic cell nuclear transfer (SCNT) to generate cloned embryos presenting an average cleavage rate exceeding 85%, and an average blastocyst formation rate exceeding 9.5%. 131 cloned embryos were transplanted into four surrogates, and all surrogates carried their pregnancies to term, and delivered 4.58% (6/131) alive cloned kittens, with 1.53% (2/131) being KIT-edited heterozygotes (KITD17/+). The KITD17/+ cats presented an obvious darkness reduction in the mackerel tabby coat. Immunohistochemical analysis (IHC) of skin tissues indicated impaired proliferation and differentiation of melanoblasts caused by the lack of exon17 in feline KIT. To our knowledge, this is the first report on coat colour modification of cats through gene editing. The findings could facilitate further understanding of the regulatory role of KIT on feline coat colour and provide a basis for the breeding of cats with commercially desired coat colour.
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Clonación de Organismos , Edición Génica , Proteínas Proto-Oncogénicas c-kit , Animales , Gatos , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Edición Génica/veterinaria , Edición Génica/métodos , Clonación de Organismos/veterinaria , Clonación de Organismos/métodos , Color del Cabello/genética , Técnicas de Transferencia Nuclear/veterinaria , FemeninoRESUMEN
As obligate parasites, viruses have evolved multiple strategies to evade the host immune defense. Manipulation of the host proteasome system to degrade specific detrimental factors is a common viral countermeasure. To identify host proteins targeted for proteasomal degradation by porcine reproductive and respiratory syndrome virus (PRRSV), we conducted a quantitative proteomics screen of PRRSV-infected Marc-145 cells under the treatment with proteasome inhibitor MG132. The data revealed that the expression levels of programmed cell death 4 (PDCD4) were strongly downregulated by PRRSV and significantly rescued by MG132. Further investigation confirmed that PRRSV infection induced the translocation of PDCD4 from the nucleus to the cytoplasm, and the viral nonstructural protein 9 (Nsp9) promoted PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway. The C-terminal domain of Nsp9 was responsible for PDCD4 degradation. As for the role of PDCD4 during PRRSV infection, we demonstrated that PDCD4 knockdown favored viral replication, while its overexpression significantly attenuated replication, suggesting that PDCD4 acts as a restriction factor for PRRSV. Mechanistically, we discovered eukaryotic translation initiation factor 4A (eIF4A) was required for PRRSV. PDCD4 interacted with eIF4A through four sites (E249, D253, D414, and D418) within its two MA3 domains, disrupting eIF4A-mediated translation initiation in the 5'-untranslated region of PRRSV, thereby inhibiting PRRSV infection. Together, our study reveals the antiviral function of PDCD4 and the viral strategy to antagonize PDCD4. These results will contribute to our understanding of the immune evasion strategies employed by PRRSV and offer valuable insights for developing new antiviral targets.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in major economic losses in the global swine industry and is difficult to control effectively. Here, using a quantitative proteomics screen, we identified programmed cell death 4 (PDCD4) as a host protein targeted for proteasomal degradation by PRRSV. We demonstrated that PDCD4 restricts PRRSV replication by interacting with eukaryotic translation initiation factor 4A, which is required for translation initiation in the viral 5'-untranslated region. Additionally, four sites within two MA3 domains of PDCD4 are identified to be responsible for its antiviral function. Conversely, PRRSV nonstructural protein 9 promotes PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway, thus weakening the anti-PRRSV function. Our work unveils PDCD4 as a previously unrecognized host restriction factor for PRRSV and reveals that PRRSV develops countermeasures to overcome PDCD4. This will provide new insights into virus-host interactions and the development of new antiviral targets.
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Proteínas Reguladoras de la Apoptosis , Factor 4A Eucariótico de Iniciación , Virus del Síndrome Respiratorio y Reproductivo Porcino , Proteínas de Unión al ARN , Proteínas no Estructurales Virales , Replicación Viral , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Animales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Porcinos , Línea Celular , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Interacciones Huésped-Patógeno , Proteolisis , Humanos , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Bone morphogenetic protein 15 (BMP15) plays a crucial role in the porcine follicular development. However, its exact functions in the in vitro maturation (IVM) of porcine oocytes remain largely unknown. Here, through cytoplasmic injection of a preassembled crRNA-tracrRNA-Cas9 ribonucleoprotein complex, we achieved BMP15 disruption in approximately 54 % of the cultured porcine oocytes. Editing BMP15 impaired the IVM of porcine oocytes, as indicated by the significantly increased abnormal spindle assembly and reduced first polar body (PB1) extrusion. The editing also impaired cytoplasmic maturation of porcine oocytes, as reflected by reduced abundant of Golgi apparatus and impaired functions of mitochondria. The impaired IVM of porcine oocytes by editing BMP15 possibly was associated with the attenuated SMAD1/5 and EGFR-ERK1/2 signaling in the cumulus granulosa cells (CGCs) and the inhibited MOS/ERK1/2 signaling in oocytes. The attenuated MOS/ERK1/2 signaling may contribute to the inactivation of maturation promoting factor (MPF) and the increased abnormal spindle assembly, leading to reduced PB1 extrusion. It also may contribute to reduced Golgi apparatus formation, and impaired functions of mitochondria. These findings expand our understanding of the regulatory role of BMP15 in the IVM of porcine oocytes and provide a basis for manipulation of porcine reproductive performance.
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Proteína Morfogenética Ósea 15 , Oocitos , Huso Acromático , Animales , Oocitos/metabolismo , Proteína Morfogenética Ósea 15/genética , Proteína Morfogenética Ósea 15/metabolismo , Porcinos , Femenino , Huso Acromático/metabolismo , Sistema de Señalización de MAP Quinasas , Mitocondrias/metabolismo , Técnicas de Maduración In Vitro de los Oocitos , Aparato de Golgi/metabolismo , Orgánulos/metabolismo , Orgánulos/genética , Transducción de SeñalRESUMEN
African swine fever virus (ASFV) and porcine reproductive and respiratory syndrome virus (PRRSV) infections lead to severe respiratory diseases in pigs, resulting in significant economic losses for the global swine industry. While numerous studies have focused on specific gene functions or pathway activities during infection, an investigation of shared immune responses in porcine alveolar macrophages (PAMs) after ASFV and PRRSV infections was lacking. In this study, we conducted a comparison using two single-cell transcriptomic datasets generated from PAMs under ASFV and PRRSV infection. Pattern recognition receptors (PRRs) RIG-I (DDX58), MDA5 (IFIH1), and LGP2 (DHX58) were identified as particularly recognizing ASFV and PRRSV, triggering cellular defense responses, including the upregulation of four cytokine families (CCL, CXCL, IL, and TNF) and the induction of pyroptosis. Through weighted gene co-expression network analysis and protein-protein interaction analysis, we identified thirteen gene and protein interactions shared by both scRNA-seq analyses, suggesting the ability to inhibit both ASFV and PRRSV viral replication. We discovered six proteins (PARP12, PARP14, HERC5, DDX60, RSAD2, and MNDA) in PAMs as inhibitors of ASFV and PRRSV replication. Collectively, our findings showed detailed characterizations of the immune responses in PAMs during ASFV and PRRSV infections, which may facilitate the treatments of these viral diseases.
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The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used to create animal models for biomedical and agricultural use owing to its low cost and easy handling. However, the occurrence of erroneous cleavage (off-targeting) may raise certain concerns for the practical application of the CRISPR-Cas9 system. In this study, we created a melanocortin 1 receptor (MC1R)-edited pig model through somatic cell nuclear transfer (SCNT) by using porcine kidney cells modified by the CRISPR-Cas9 system. We then carried out whole-genome sequencing of two MC1R-edited pigs and two cloned wild-type siblings, together with the donor cells, to assess the genome-wide presence of single-nucleotide variants and small insertions and deletions (indels) and found only one candidate off-target indel in both MC1R-edited pigs. In summary, our study indicates that the minimal off-targeting effect induced by CRISPR-Cas9 may not be a major concern in gene-edited pigs created by SCNT.
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Sistemas CRISPR-Cas , Receptor de Melanocortina Tipo 1 , Animales , Porcinos/genética , Receptor de Melanocortina Tipo 1/genética , Sistemas CRISPR-Cas/genética , Edición Génica , Mutación , Mutación INDEL/genéticaRESUMEN
Structural variations have emerged as an important driving force for genome evolution and phenotypic variation in various organisms, yet their contributions to genetic diversity and adaptation in domesticated animals remain largely unknown. Here we constructed a pangenome based on 250 sequenced individuals from 32 pig breeds in Eurasia and systematically characterized coding sequence presence/absence variations (PAVs) within pigs. We identified 308.3-Mb nonreference sequences and 3438 novel genes absent from the current reference genome. Gene PAV analysis showed that 16.8% of the genes in the pangene catalog undergo PAV. A number of newly identified dispensable genes showed close associations with adaptation. For instance, several novel swine leukocyte antigen (SLA) genes discovered in nonreference sequences potentially participate in immune responses to productive and respiratory syndrome virus (PRRSV) infection. We delineated previously unidentified features of the pig mobilome that contained 490,480 transposable element insertion polymorphisms (TIPs) resulting from recent mobilization of 970 TE families, and investigated their population dynamics along with influences on population differentiation and gene expression. In addition, several candidate adaptive TE insertions were detected to be co-opted into genes responsible for responses to hypoxia, skeletal development, regulation of heart contraction, and neuronal cell development, likely contributing to local adaptation of Tibetan wild boars. These findings enhance our understanding on hidden layers of the genetic diversity in pigs and provide novel insights into the role of SVs in the evolutionary adaptation of mammals.
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Cruzamiento , Genoma , Humanos , Animales , Porcinos , Variación Genética , MamíferosRESUMEN
BACKGROUND: Chinese indigenous pigs are popular with consumers for their juiciness, flavour and meat quality, but they have lower meat production. Insulin-like growth factor 2 (IGF2) is a maternally imprinted growth factor that promotes skeletal muscle growth by regulating cell proliferation and differentiation. A single nucleotide polymorphism (SNP) within intron 3 of porcine IGF2 disrupts a binding site for the repressor, zinc finger BED-type containing 6 (ZBED6), leading to up-regulation of IGF2 and causing major effects on muscle growth, heart size, and backfat thickness. This favorable mutation is common in Western commercial pig populations, but absent in most Chinese indigenous pig breeds. To improve meat production of Chinese indigenous pigs, we used cytosine base editor 3 (CBE3) to introduce IGF2-intron3-C3071T mutation into porcine embryonic fibroblasts (PEFs) isolated from a male Liang Guang Small Spotted pig (LGSS), and single-cell clones harboring the desired mutation were selected for somatic cell nuclear transfer (SCNT) to generate the founder line of IGF2T/T pigs. RESULTS: We found the heterozygous progeny IGF2C/T pigs exhibited enhanced expression of IGF2, increased lean meat by 18%-36%, enlarged loin muscle area by 3%-17%, improved intramuscular fat (IMF) content by 18%-39%, marbling score by 0.75-1, meat color score by 0.53-1.25, and reduced backfat thickness by 5%-16%. The enhanced accumulation of intramuscular fat in IGF2C/T pigs was identified to be regulated by the PI3K-AKT/AMPK pathway, which activated SREBP1 to promote adipogenesis. CONCLUSIONS: We demonstrated the introduction of IGF2-intron3-C3071T in Chinese LGSS can improve both meat production and quality, and first identified the regulation of IMF deposition by IGF2 through SREBP1 via the PI3K-AKT/AMPK signaling pathways. Our study provides a further understanding of the biological functions of IGF2 and an example for improving porcine economic traits through precise base editing.
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Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell proliferation, apoptosis, and differentiation. Our previous study showed that KLF4 expression is upregulated in skeletal muscle ontogeny during embryonic development in pigs, suggesting its importance for skeletal muscle development and muscle function. We revealed here that KLF4 plays a critical role in skeletal muscle development and regeneration. Specific knockout of KLF4 in skeletal muscle impaired muscle formation further affecting physical activity and also defected skeletal muscle regeneration. In vitro, KLF4 was highly expressed in proliferating myoblasts and early differentiated cells. KLF4 knockdown promoted myoblast proliferation and inhibited myoblast fusion, while its overexpression showed opposite results. Mechanically, in proliferating myoblasts, KLF4 inhibits myoblast proliferation through regulating cell cycle arrest protein P57 by directly targeting its promoter; while in differentiated myoblasts, KLF4 promotes myoblast fusion by transcriptionally activating Myomixer. Our study provides mechanistic information for skeletal muscle development, reduced muscle strength and impaired regeneration after injury and unveiling the mechanism of KLF4 in myogenic regulation.
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Factor 4 Similar a Kruppel , Desarrollo de Músculos , Femenino , Embarazo , Animales , Porcinos , Desarrollo de Músculos/genética , Diferenciación Celular/genética , Apoptosis , Proteínas de Ciclo Celular , Músculo EsqueléticoRESUMEN
In brief: The regulatory role of BMP15 on porcine ovarian follicular development still remains unclear. This study reveals that biallelic editing of BMP15 impairs SMAD signaling and inhibits granulosa cell proliferation, resulting in porcine follicular development arrest and ovarian hypoplasia. Abstract: Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta (TGF-ß) superfamily, which is critical for facilitating ovarian folliculogenesis in mono-ovulatory mammalian species but is not essential in polyovulatory mice. Our previously established BMP15-edited pigs presented varied female reproductive phenotypes, suggesting the important role of BMP15 in ovarian folliculogenesis in polyovulatory pigs. To understand the regulatory mechanism underlying the effect of BMP15 on porcine ovarian follicular development, we molecularly characterized infertile biallelic-BMP15-edited gilts with ovarian hypoplasia. We found that an absence of BMP15 proteins in biallelic-BMP15-edited gilts can lead to premature activation of primordial follicles, possibly through the upregulation of KITLG-KIT-PI3K-AKT signaling pathways. However, this absence severely impaired SMAD (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling, causing severely reduced granulosa cell proliferation, leading to the arrest of follicular development during the preantral stage and ovarian hypoplasia, resulting in complete infertility. Our study expands the understanding of the molecular functions of BMP15 in nonrodent polyovulatory mammals.
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Proteína Morfogenética Ósea 15 , Fosfatidilinositol 3-Quinasas , Femenino , Porcinos , Animales , Ratones , Proteína Morfogenética Ósea 15/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Folículo Ovárico/metabolismo , Ovario/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor 9 de Diferenciación de Crecimiento/genética , Mamíferos/metabolismoRESUMEN
E3 ubiquitin ligases are closely related to cell division, differentiation, and survival in all eukaryotes and play crucial regulatory roles in multiple biological processes and diseases. While Deltex2, as a member of the DELTEX family ubiquitin ligases, is characterized by a RING domain followed by a C-terminal domain (DTC), its functions and underlying mechanisms in myogenesis have not been fully elucidated. Here, we report that Deltex2, which is highly expressed in muscles, positively regulates myoblast proliferation via mediating the expression of Pax7. Meanwhile, we find that Deltex2 is translocated from the nucleus into the cytoplasm during myogenic differentiation, and further disclose that Deltex2 inhibits myoblast differentiation and interacts with MyoD, resulting in the ubiquitination and degradation of MyoD. Altogether, our findings reveal the physiological function of Deltex2 in orchestrating myogenesis and delineate the novel role of Deltex2 as a negative regulator of MyoD protein stability.
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Fenómenos Biológicos , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Diferenciación Celular , Ubiquitina/metabolismo , Mioblastos/metabolismo , Proliferación CelularRESUMEN
BACKGROUND: Skeletal muscle development is a multistep process whose understanding is central in a broad range of fields and applications, from the potential medical value to human society, to its economic value associated with improvement of agricultural animals. Skeletal muscle initiates in the somites, with muscle precursor cells generated in the dermomyotome and dermomyotome-derived myotome before muscle differentiation ensues, a developmentally regulated process that is well characterized in model organisms. However, the regulation of skeletal muscle ontogeny during embryonic development remains poorly defined in farm animals, for instance in pig. Here, we profiled gene expression and chromatin accessibility in developing pig somites and myotomes at single-cell resolution. RESULTS: We identified myogenic cells and other cell types and constructed a differentiation trajectory of pig skeletal muscle ontogeny. Along this trajectory, the dynamic changes in gene expression and chromatin accessibility coincided with the activities of distinct cell type-specific transcription factors. Some novel genes upregulated along the differentiation trajectory showed higher expression levels in muscular dystrophy mice than that in healthy mice, suggesting their involvement in myogenesis. Integrative analysis of chromatin accessibility, gene expression data, and in vitro experiments identified EGR1 and RHOB as critical regulators of pig embryonic myogenesis. CONCLUSIONS: Collectively, our results enhance our understanding of the molecular and cellular dynamics in pig embryonic myogenesis and offer a high-quality resource for the further study of pig skeletal muscle development and human muscle disease.
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Secuenciación de Inmunoprecipitación de Cromatina , Análisis de Expresión Génica de una Sola Célula , Animales , Ratones , Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Análisis de la Célula Individual , PorcinosRESUMEN
Improving the prediction accuracies of economically important traits in genomic selection (GS) is a main objective for researchers and breeders in the livestock industry. This study aims at utilizing potentially functional SNPs and QTLs identified with various genome-wide association study (GWAS) models in GS of pig growth traits. We used three well-established GWAS methods, including the mixed linear model, Bayesian model and meta-analysis, as well as 60K SNP-chip and whole genome sequence (WGS) data from 1734 Yorkshire and 1123 Landrace pigs to detect SNPs related to four growth traits: average daily gain, backfat thickness, body weight and birth weight. A total of 1485 significant loci and 24 candidate genes which are involved in skeletal muscle development, fatty deposition, lipid metabolism and insulin resistance were identified. Compared with using all SNP-chip data, GS with the pre-selected functional SNPs in the standard genomic best linear unbiased prediction (GBLUP), and a two-kernel based GBLUP model yielded average gains in accuracy by 4 to 46% (from 0.19 ± 0.07 to 0.56 ± 0.07) and 5 to 27% (from 0.16 ± 0.06 to 0.57 ± 0.05) for the four traits, respectively, suggesting that the prioritization of preselected functional markers in GS models had the potential to improve prediction accuracies for certain traits in livestock breeding.
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Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/µL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/µL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.
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Proteína Morfogenética Ósea 15 , Oocitos , Porcinos , Femenino , Animales , Proteína Morfogenética Ósea 15/genética , Microinyecciones/veterinaria , Oocitos/fisiología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Células del Cúmulo/fisiología , MamíferosRESUMEN
Quercetin (QUE) is a component of the flavonoid family that shows various therapeutic properties, such as antioxidant effects. However, whether QUE affects porcine oocyte in vitro aging has not yet been investigated. Therefore, in this study, we applied various doses of QUE to freshly isolated porcine oocytes and found that 10 µM QUE improved the oocyte maturation rate in vitro, as reflected by the increased degree of cumulus cell expansion and first polar body extrusion. More importantly, we found that QUE reduced in vitro aging and improved the maturity level of porcine oocytes after another 24 h of culturing, accompanied by the upregulated expression levels of bone morphogenetic protein 15, growth differentiation factor 9, Moloney sarcoma oncogene, and cyclin-dependent kinase 2. In addition, we found that QUE treatment significantly reduced the intracellular reactive oxygen species levels, apoptosis, and autophagy and upregulated the expression levels of superoxide dismutase 2 and catalase in aged porcine oocytes. In addition, QUE restored impaired mitochondrial membrane potential and spindle assembly in aged porcine oocytes. Our findings demonstrate that QUE can protect porcine oocytes from in vitro aging by reducing oxidative stress and maintaining mitochondrial function.
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Porcine reproductive and respiratory syndrome viruses (PRRSVs) pose a serious threat to the swine industry in China, which has caused great difficulties for porcine reproductive and respiratory syndrome (PRRS) immune prevention and control, due to its easily mutable and recombinant nature. In this study, two novel PRRSV strains, which were named GD-H1 and GD-F1, were isolated and fully sequenced from pig farms in Guangdong province, China. The phylogenetic analysis and recombination analysis revealed that the GD-H1 and GD-F1 were generated by the recombination of NADC30-like and NADC34-like strains which were different from the previously prevalent strain. Further pathogenic studies on piglets and sows found that the recombinant strains could cause piglets high fever, loss of appetite and lung lesions, but no piglets died. However, the recombinant strains could cause acute death and abortion in pregnant sow infection models together with average survival rates of 62.5% and 37.5% abortion rates, respectively. These findings indicated that the recombinant strains were extremely pathogenic to sows. Therefore, we report two clinical novel recombinant strains of PRRSV that are different from the traditional epidemic strains in China, which may provide early warning and support for PRRS immune prevention and control.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Virulencia , Filogenia , Genoma Viral , China/epidemiología , Variación GenéticaRESUMEN
The white coat color of Yorkshire pigs is caused by the dominant white I allele, which has been associated with at least one copy of the 450-kb duplication encompassing the entire KIT gene and a splice mutation (G > A) at the first base of intron 17. The splice mutation in KIT has an adverse effect on pigmentation in mice. Therefore, removing the 450 kb duplications harboring the KIT copy with splice mutations is expected to affect Yorkshire pig pigmentation. In this study, we describe the use of a Yorkshire pig kidney cell strain with the I?/IBe-ed genotype, previously created by CRISPR-Cas9, as donor cells for somatic cell nuclear transfer to generate gene-edited Yorkshire pigs. The removal of the 450 kb duplications harboring the KIT copy with splice mutation did not alter the white coat color of Yorkshire pigs, which was confirmed by the absence of fully mature melanocytes and melanin accumulation in the hair follicles. Except for the improved transcription of tyrosinase, and slight increase in microphthalmia transcription factor and tyrosinase-related protein 1 protein expression, there was no significant impact of the removal of splice mutations on genes and signaling pathways (PI3K/AKT) involved in melanogenesis. However, the removal of the 450 kb duplications harboring the KIT copy with splice mutation substantially improved fresh meat color accompanied by significantly increased red blood cell number, which merits further investigation. Our study provides new insights into the role of structural mutations of the KIT gene in the formation of white coat color and erythropoiesis in Yorkshire pigs.
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Color del Cabello , Proteínas Proto-Oncogénicas c-kit , Animales , Sistemas CRISPR-Cas/genética , Edición Génica , Color del Cabello/genética , Monofenol Monooxigenasa/genética , Fosfatidilinositol 3-Quinasas/genética , Pigmentación/genética , Proteínas Proto-Oncogénicas c-kit/genética , Porcinos/genética , Carne de CerdoRESUMEN
Protein arginine methyltransferase 1 (PRMT1) methylates a variety of histone and nonhistone protein substrates to regulate multiple cellular functions such as transcription, DNA damage response, and signal transduction. It has been reported as an emerging regulator of various metabolic pathways including glucose metabolism in the liver, atrophy in the skeletal muscle, and lipid catabolism in the adipose tissue. However, the underlying mechanisms governing how PRMT1 regulates adipogenesis remain elusive. Here, we delineate the roles of PRMT1 in mitotic clonal expansion and adipocyte differentiation. Gain and loss of functions demonstrate that PRMT1 is essential for adipogenesis of 3T3-L1 and C3H10T1/2 cells. Mechanistically, we show PRMT1 promotes the expression of transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) by catalyzing histone modification H4R3me2a and impedes the activation of Wnt/ß-catenin signaling by increasing the level of Axin to accelerate adipogenic differentiation. In addition, we demonstrate mitotic clonal expansion is suppressed by PRMT1 deficiency. PRMT1 interacts with transcription factor CCATT enhancer-binding protein ß (C/EBPß), and the absence of PRMT1 leads to the depressed phosphorylation of C/EBPß. Interestingly, we discover PRMT1 acts as a positive regulator of C/EBPß protein stability through decreasing the level of E3 ubiquitin ligase Smurf2, which promotes the ubiquitination and degradation of C/EBPß, thus facilitating adipogenesis. Collectively, these discoveries highlight a critical role of PRMT1 in adipogenesis and provide potential therapeutic targets for the treatment of obesity.