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BACKGROUND: Nasal obstruction in humans leads to mouth breathing and subsequent hypoxia in the entire body. Furthermore, nasal obstruction in growing children affects craniofacial growth and development. OBJECTIVE: To investigate the effects of unilateral nasal obstruction (UNO) on craniofacial growth in mice of different ages, particularly on the morphology of the nasomaxillary complex and mandible. METHODS: Mice aged 3, 6 and 12 weeks were selected as representatives of juvenile, adolescent and adult stages, respectively. A total of 30 male C57BL/6J mice (10 mice each at the ages of 3, 6 and 12 weeks) were used in this study for a 3-week experiment. The mice in each age stage were randomly and evenly assigned to either the control group (C3+3, C6+3 and C12+3) or the experimental group (E3+3, E6+3 and E12+3). The UNO model in experimental group was constructed by plugging the mouse's left nostril, thereby disrupting its normal nasal breathing pattern and inducing hypoxia. The control group underwent the sham procedure. After 3 weeks, the length, width and height of the cranium, nasomaxillary complex and mandible of each group were measured on two-dimensional images constructed by micro-computed tomography. Furthermore, the impact of UNO on mouse growth was evaluated through the measurement of femoral length. RESULTS: In juvenile mice, UNO inhibited the growth of cranial width, cranial height and mandibular length. In adolescent mice, UNO impeded the growth of the femoral length, cranial length, nasomaxillary length and mandibular length. In adult mice, no significant negative effects of UNO on craniofacial growth were found. CONCLUSION: Referring to the experimental results, in addition to actively treating nasal obstruction in patients, it is important to monitor the growth of the mandible in juveniles, as well as the nasomaxillary and mandibular growth in adolescents during orthodontic clinical practice.
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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.
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Avanço Mandibular , Osteogênese , Masculino , Animais , Camundongos , Camundongos Endogâmicos C57BL , Côndilo Mandibular , Angiogênese , Microtomografia por Raio-X , Imunoglobulina GRESUMO
Clinical investigations have highlighted disruptions in bone metabolic processes and abnormal fluctuations in serum indicator levels during the onset of leg disease (LD) in broilers. However, the presence of a genetic causal relationship for this association remains undetermined. Therefore, the aim of this study is to discern the risk factors underlying LD development using 1235 sequenced white-feathered broilers. We employed Mendelian randomization (MR) analysis to assess the associations of bone strength (BS), bone mineral density (BMD), tibial bone weight (TBW), tibial bone length (TBL), tibial bone diameter (TBD), bone ash (BA), ash calcium (Ash Ca), ash phosphorus (Ash P), serum calcium (Ca), serum phosphorus (P), serum alkaline phosphatase (ALP), and serum osteoprotegerin (OPG) with the incidence of LD. Compelling evidence underscores a causal link between the risk of developing LD and decreased BMD (odds ratio (OR) = 0.998; 95% CI: 0.983, 0.993; P < 0.001) and narrower TBD (OR = 0.985, 95% CI: 0.975, 0.994, P = 0.002). Additionally, serum OPG concentrations (OR: 0.995, 95% CI: 0.992, 0.999, P = 0.008) were associated with BMD (OR = 0.0078, 95% CI = 0.0043 to 0.0140, P < 0.001), indicating a robust genetic relationship between ALP concentrations (OR: 0.988, 95% CI: 0.984, 0.993, P < 0.001) and TBD (OR = 0.0046, 95% CI = 0.0026, 0.0083, P < 0.001). Moreover, elevated serum Ca (OR: 0.564, 95% CI: 0.487, 0.655, P < 0.001) and P (OR: 0.614, 95% CI: 0.539, 0.699, P < 0.001) levels were associated with a narrower TBD. Elevated serum levels of Ca, P, ALP, and OPG contribute to disturbances in bone metabolism, while decreased BMD and narrower TBD are associated with a greater risk of developing LD in broilers. This discovery elucidates the metabolic risk factors for LD in broilers and could provide information on LDs, such as osteoporosis, in humans.
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Densidade Óssea , Galinhas , Análise da Randomização Mendeliana , Animais , Galinhas/genética , Fatores de Risco , Densidade Óssea/genética , Predisposição Genética para Doença , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/epidemiologia , Osteoprotegerina/genética , Osteoprotegerina/sangue , Polimorfismo de Nucleotídeo ÚnicoRESUMO
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.
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MicroRNAs , Ovinos/genética , Feminino , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Perfilação da Expressão Gênica , RNA Mensageiro/genética , Fertilidade/genética , Endométrio/metabolismo , Mamíferos/genéticaRESUMO
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.
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Avanço Mandibular , Osteogênese , Masculino , Camundongos , Animais , Microtomografia por Raio-X , Camundongos Endogâmicos C57BL , Côndilo Mandibular/diagnóstico por imagemRESUMO
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.
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Avanço Mandibular , Côndilo Mandibular , Masculino , Camundongos , Animais , Côndilo Mandibular/diagnóstico por imagem , Côndilo Mandibular/patologia , Osteogênese/fisiologia , Desferroxamina/farmacologia , Microtomografia por Raio-X , Camundongos Endogâmicos C57BLRESUMO
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.
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RNA Circular/genética , Análise de Sequência de RNA/veterinária , Lã/crescimento & desenvolvimento , Animais , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Ovinos , Pele , Lã/químicaRESUMO
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.
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Ácido Clorogênico/farmacologia , Eucommiaceae/química , Carne/normas , Extratos Vegetais/farmacologia , Folhas de Planta/química , Ração Animal/análise , Animais , Ácido Clorogênico/química , Óleo de Milho/química , Dieta/veterinária , Oxirredução , Extratos Vegetais/química , Suínos/crescimento & desenvolvimentoRESUMO
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.
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Má Oclusão Classe II de Angle , Aparelhos Ortodônticos Funcionais , Cefalometria , Feminino , Humanos , Masculino , Mandíbula , RadiografiaRESUMO
BACKGROUND: Sheep are valuable resources for the animal fibre industry. Therefore, identifying genes which regulate wool growth would offer strategies for improving the quality of fine wool. In this study, we employed Agilent sheep gene expression microarray and proteomic technology to compare the gene expression patterns of the body side (hair-rich) and groin (hairless) skins of Aohan fine wool sheep (a Chinese indigenous breed). RESULTS: Comparing the body side to the groin skins (S/G) of Aohan fine wool sheep, the microarray study revealed that 1494 probes were differentially expressed, including 602 more highly expressed and 892 less highly expressed probes. The microarray results were verified by means of quantitative PCR. Cluster analysis could distinguish the body side skin and the groin skin. Based on the Database for Annotation, Visualization and Integrated Discovery (DAVID), 38 of the differentially expressed genes were classified into four categories, namely regulation of receptor binding, multicellular organismal process, protein binding and macromolecular complex. Proteomic study revealed that 187 protein spots showed significant (p < 0.05) differences in their respective expression levels. Among them, 46 protein entries were further identified by MALDI-TOF/MS analyses. CONCLUSIONS: Microarray analysis revealed thousands of differentially expressed genes, many of which were possibly associated with wool growth. Several potential gene families might participate in hair growth regulation. Proteomic analysis also indentified hundreds of differentially expressed proteins.
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Carneiro Doméstico/genética , Pele/metabolismo , Lã/crescimento & desenvolvimento , Animais , Feminino , Regulação da Expressão Gênica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Carneiro Doméstico/crescimento & desenvolvimento , TranscriptomaRESUMO
Sheep are valuable resources for the wool industry. Wool growth of Aohan fine wool sheep has cycled during different seasons in 1 year. Therefore, identifying genes that control wool growth cycling might lead to ways for improving the quality and yield of fine wool. In this study, we employed Agilent sheep gene expression microarray and proteomic technology to compare the gene expression patterns of the body side skins at August and December time points in Aohan fine wool sheep (a Chinese indigenous breed). Microarray study revealed that 2,223 transcripts were differentially expressed, including 1,162 up-regulated and 1,061 down-regulated transcripts, comparing body side skin at the August time point to the December one (A/D) in Aohan fine wool sheep. Then seven differentially expressed genes were selected to validated the reliability of the gene chip data. The majority of the genes possibly related to follicle development and wool growth could be assigned into the categories including regulation of receptor binding, extracellular region, protein binding and extracellular space. Proteomic study revealed that 84 protein spots showed significant differences in expression levels. Of the 84, 63 protein spots were upregulated and 21 were downregulated in A/D. Finally, 55 protein points were determined through MALDI-TOF/MS analyses. Furthermore, the regulation mechanism of hair follicle might resemble that of fetation.
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Folículo Piloso/metabolismo , Ovinos/genética , Lã/crescimento & desenvolvimento , Animais , Eletroforese em Gel Bidimensional , Feminino , Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteômica , RNA/genética , Reprodutibilidade dos Testes , TranscriptomaRESUMO
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.
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Sine oculis homeobox 1 (Six1) homeodomain transcription factor is implicated in the genesis of muscle fiber type diversity, but its regulatory mechanisms on the formation of muscle fiber type are still poorly understood. To elucidate the biological roles of Six1 gene in muscle fiber formation, we established C2C12 cell line overexpressing Six1 and determined the effects of forced Six1 expression on muscle-specific genes expression, cell proliferation, and cell cycles. Our results indicated that Six1 overexpression could significantly promote the expression of fast-type muscle genes Atp2a1, Srl, and Mylpf. Furthermore, Six1 overexpressing C2C12 cells displayed a relative lower proliferative potential, and cell cycle analysis showed that Six1 exerted its role in cell cycle primarily through the regulation of G1/S and G2/M phases. In conclusion, Six1 plays an essential role in modulation of the fast-twitch muscle fiber phenotype through up-regulating fast-type muscle genes expression, and it could suppress the proliferation of muscle cells.
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Proliferação de Células , Proteínas de Homeodomínio/genética , Fibras Musculares de Contração Rápida/metabolismo , Mioblastos/metabolismo , Animais , Western Blotting , Ciclo Celular/genética , Linhagem Celular , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fibras Musculares de Contração Rápida/citologia , Mioblastos/citologia , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de TempoRESUMO
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.
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Remodelação Óssea , Técnicas de Movimentação Dentária , Aceleração , Adulto , Citocinas , HumanosRESUMO
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.
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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).
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Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes , Genes Reporter , Luciferases/metabolismo , Mamíferos/metabolismo , Plasmídeos/genética , Animais , Sequência de Bases , Linhagem Celular , DNA/metabolismo , Reparo do DNA , Vetores Genéticos/metabolismo , Luciferases/genética , Oligonucleotídeos/metabolismo , RNA Guia de Cinetoplastídeos/genética , Reprodutibilidade dos Testes , Ovinos , Transformação GenéticaRESUMO
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.
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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.
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Proteômica , Carneiro Doméstico/genética , Transcriptoma/genética , Fibra de Lã , Animais , Cruzamento , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Pele/metabolismo , Lã/química , Lã/metabolismoRESUMO
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.