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A white ear lobe chicken embryo (WELCE) fibroblast cell bank, containing 322 tubes of frozen cells, was successfully established from primary explants of 57 embryo samples. The cells were morphologically consistent with fibroblasts, and the growth curve was sigmoidal with a population doubling time (PDT) of 48 h. Karyotyping and G-banding indicated a total chromosome number of 2n=78; the rate of diploidy in the cell bank was 97.62%. The cells were also free from bacterial, fungal, viral and mycoplasma contamination. Analysis of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzymes ruled out cross-contamination between cells. In order to study exogenous gene expression, six fluorescent proteins were transfected into the WELCE cells. The transfection efficiency of these genes was between 10.1 and 41.9%. The corresponding fluorescence was distributed throughout the cytoplasm and nucleus 24h after transfection. The results indicate that the quality of the cell line meet the quality requirements of the ATCC (American Type Culture Collection).

Simple Summary: In our research, candidate genes related to sheep-milk production were revealed by a genome-resequencing analysis and a genome-signal-selection analysis, and a RT-qPCR experiment was performed to prove the expression levels of these candidate genes, the results showed that the FCGR3A gene's expression level had a significant negative relationship with sheep-milk production. Natural selection and domestication have shaped modern sheep populations into a vast range of phenotypically diverse breeds. Among these breeds, dairy sheep have a smaller population than meat sheep and wool sheep, and less research is performed on them, but the lactation mechanism in dairy sheep is critically important for improving animal-production methods. In this study, whole-genome sequences were generated from 10 sheep breeds, including 57 high-milk-yield sheep and 44 low-milk-yield sheep, to investigate the genetic signatures of milk production in dairy sheep, and 59,864,820 valid SNPs (Single Nucleotide Polymorphisms) were kept after quality control to perform population-genetic-structure analyses, gene-detection analyses, and gene-function-validation analyses. For the population-genetic-structure analyses, we carried out PCA (Principal Component Analysis), as well as neighbor-joining tree and structure analyses to classify different sheep populations. The sheep used in our study were well distributed in ten groups, with the high-milk-yield-group populations close to each other and the low-milk-yield-group populations showing similar classifications. To perform an exact signal-selection analysis, we used three different methods to find SNPs to perform gene-annotation analyses within the 995 common regions derived from the fixation index (FST), nucleotide diversity (Ɵπ), and heterozygosity rate (ZHp) results. In total, we found 553 genes that were located in these regions. These genes mainly participate in the protein-binding pathway and the nucleoplasm-interaction pathway, as revealed by the GO- and KEGG-function-enrichment analyses. After the gene selection and function analyses, we found that FCGR3A, CTSK, CTSS, ARNT, GHR, SLC29A4, ROR1, and TNRC18 were potentially related to sheep-milk-production traits. We chose the strongly selected genes, FCGR3A, CTSK, CTSS, and ARNT during the signal-selection analysis to perform a RT-qPCR (Reale time Quantitative Polymerase Chain Reaction) experiment to validate their expression-level relationship with milk production, and the results showed that FCGR3A has a significant negative relationship with sheep-milk production, while other three genes did not show any positive or negative relations. In this study, it was discovered and proven that the candidate gene FCGR3A potentially contributes to the milk production of dairy sheep and a basis was laid for the further study of the genetic mechanism underlying the strong milk-production traits of sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: A unique structure of ruminants, the horn trait is not only closely related to natural and sexual selection but is also an important trait for polled sheep breeding. RXFP2 may be a crucial gene in regulating sheep horn. However, the underlying genetic mechanisms of sheep horn development remain largely unknown. In this study, we investigated the gene expression profile of the horn buds in sheep fetuses using RNA-seq technology. We identified 68 differentially expressed genes in the horn buds of 105-day-old Altay sheep fetuses, including RXFP2, FOXL2, and TNN. Further, we found that the Wnt signaling pathway may be responsible for horn development. Our study provides new possible marker genes for horn development, which may promote our understanding of the genetic mechanisms of sheep horn formation. Horns, also known as headgear, are a unique structure of ruminants. As ruminants are globally distributed, the study of horn formation is critical not only for increasing our understanding of natural and sexual selection but also for the breeding of polled sheep breeds to facilitate modern sheep farming. Despite this, a significant number of the underlying genetic pathways in sheep horn remain unclear. In this study, to clarify the gene expression profile of horn buds and investigate the key genes in horn bud formation, RNA-sequencing (RNA-seq) technology was utilized to investigate differential gene expression in the horn buds and adjacent forehead skin of Altay sheep fetuses. There were only 68 differentially expressed genes (DEGs) identified, consisting of 58 up-regulated genes and 10 down-regulated genes. RXFP2 was differentially up-regulated in the horn buds and had the highest significance (p-value = 7.42 × 10−14). In addition, 32 DEGs were horn-related genes identified in previous studies, such as RXFP2, FOXL2, SFRP4, SFRP2, KRT1, KRT10, WNT7B, and WNT3. Further, Gene Ontology (GO) analysis showed that the DEGs were mainly enriched with regard to growth, development, and cell differentiation. Pathway analysis revealed that the Wnt signaling pathway may be responsible for horn development. Further, through combining the protein–protein interaction networks of the DEGs, it was found that the top five hub genes, namely, ACAN, SFRP2, SFRP4, WNT3, and WNT7B, were also associated with horn development. Our results suggest that only a few key genes, including RXFP2, are involved in bud formation. This study not only validates the expression of candidate genes identified at the transcriptome level in previous studies but also provides new possible marker genes for horn development, which may promote our understanding of the genetic mechanisms of horn formation. [ABSTRACT FROM AUTHOR]

Meanwhile, we integrated the gene sets (Table S3) and compared them with the DEGs identified in this study; these sets included the horn-related genes and proteins of sheep, goats, and cattle horns identified in previous studies [1,3,10,12-15,27-34]. Meanwhile, we integrated the gene sets (Table S3) and compared them with the DEGs identified in this study; these sets included the horn-related genes and proteins of sheep, goats, and cattle horns identified in previous studies [1,3,10,12-15,17,29-35]. The shape of sheep horn buds is similar to that of cattle horn buds [20]. [Extracted from the article]

The FGF5 gene has been associated with the regulation of fibre length in mammals, including cashmere goats. A deletion variant at ~14 kb downstream of the FGF5 gene showed significant divergence between cashmere and non‐cashmere goats in previous studies. In this study, we designed specific primers to genotype the deletion variant. The results of gel electrophoresis and Sanger sequencing revealed that a 507‐bp deletion mutation is located at 95 454 685–95 455 191 of chromosome 6 in goats. Genotyping data from a large panel of 288 goats showed that the deletion at the FGF5 gene locus appeared to be associated with cashmere length. The deletion variant was close to fixation (frequency 0.97) in cashmere goats. Furthermore, electrophoretic mobility shift assays for evaluating DNA–protein interaction and mRNA expression levels of FGF5 suggested that the deletion variant may serve as a cis‐acting element by specifically binding transcription factors to mediate quantitative changes in FGF5 mRNA expression. Our study illustrates how a structural mutation of the FGF5 gene has contributed to the cashmere growth phenotype in domestic goats. The deletion mutation within the FGF5 gene could potentially serve as a molecular marker of cashmere growth in cashmere goat breeding. [ABSTRACT FROM AUTHOR]

Bactrian camels (Camelus bactrianus) are highly adapted to the desert and semi-desert environments of Asia and developed unique physiological adaptations to cold, heat, drought, and nutrient-poor conditions. These animals are an ideal model for studying desert adaptation. However, the transcriptome of different Bactrian camel tissues has not been profiled. This study performed a comprehensive transcriptome analysis of nine fetal and adult tissues. A total of 20,417 coding genes were identified, and 2.4 billion reads were generated. Gene expression and functional analyses revealed that approximately 50% of the identified genes were ubiquitously expressed, and one-third were tissue-elevated genes, which were enriched in pathways related to the biological functions of the corresponding tissue. Weighted gene co-expressed network analysis (WGCNA) identified four modules—fat metabolism, water balance, immunity, and digestion—and several hub genes, including APOA1, TMEM174, CXCL12, and MYL9. The analysis of differentially expressed genes (DEGs) between fetal and adult tissues revealed that downregulated genes were enriched in tissue development, whereas upregulated genes were enriched in biological function in adult camels. DEGs in the hump were enriched in immune-related pathways, suggesting that this tissue is involved in immunity. This study is the first to generate a transcriptome atlas of major tissues in Bactrian camels and explores the genes potentially involved in the adaptation to desert environments. [ABSTRACT FROM AUTHOR]

ZBED6 (zinc finger BED domain containing protein 6) is a transcription factor unique to placental mammals and its interaction with the IGF2 (insulin-like growth factor 2) locus plays a prominent role in the regulation of postnatal skeletal muscle growth. Here, we generated lean Bama miniature pigs by generating ZBED6-knockout (ZBED6−/−) and investigated the mechanism underlying ZBED6 in growth of muscle and internal organs of placental mammals. ZBED6−/− pigs show markedly higher lean mass, lean mass rate, larger muscle fiber area and heavier internal organs (heart and liver) than wild-type (WT) pigs. The striking phenotypic changes of ZBED6-/- pigs coincided with remarkable upregulation of IGF2 mRNA and protein expression across three tissues (gastrocnemius muscle, longissimus dorsi, heart). Despite a significant increase in liver weight, ZBED6-/- pigs show comparable levels of IGF2 expression to those of WT controls. A mechanistic study revealed that elevated methylation in the liver abrogates ZBED6 binding at the IGF2 locus, explaining the unaltered hepatic IGF2 expression in ZBED6-/- pigs. These results indicate that a ZBED6-IGF2-independent regulatory pathway exists in the liver. Transcriptome analysis and ChIP-PCR revealed new ZBED6 target genes other than IGF2, including cyclin dependent kinase inhibitor 1A (CDKN1A) and tsukushi, small leucine rich proteoglycan (TSKU), that regulates growth of muscle and liver, respectively. Author summary: The lean meat rate is an important economic trait for the swine industry and it is determined by muscle growth and development. A single base change in intron 3 of the insulin-like growth factor 2 (IGF2) gene increases meat production in pigs by disrupting a binding site for zinc finger BED domain containing protein 6 (ZBED6). Chinese indigenous pig breeds carrying the homozygous IGF2 wild-type allele produce low lean meat. We thus generate a lean pig model in Chinese Bama pig by knocking out ZBED6. In this model, we demonstrate that ZBED6 KO increases muscle and internal organ growth through ZBED6-IGF2 axis and other target genes. These results not only open new strategies for lean meat breeding in Chinese indigenous pigs, but also provide new insights to the global function of ZBED6 in organ growth and development. [ABSTRACT FROM AUTHOR]

Background: Long intergenic noncoding RNAs (lincRNAs) have been recognized in recent years as key regulators of biological processes. However, lincRNAs in goat remain poorly characterized both across various tissues and during different developmental stages in goat (Capra hircus). Results: We performed the genome-wide discovery of the lincRNAs in goat by combining the RNA-seq dataset that were generated from 28 cashmere goat skin samples and the 12 datasets of goat tissues downloaded from the NCBI database. We identified a total of 5546 potential lincRNA transcripts that overlapped 3641 lincRNA genes. These lincRNAs exhibited a tissue-specific pattern. Specifically, there are 584 lincRNAs expressed exclusively in only one tissue, and 91 were highly expressed in hair follicle (HF). In addition, 2350 protein-coding genes and 492 lincRNAs were differentially expressed in the skin of goat. The majority exhibited the remarkable differential expression during the transition of the goat skin from the May–June to August–October time point, which covered the different seasons. Fundamental biological processes, such as skin development, were significantly enriched in these genes. Furthermore, we identified several lincRNAs highly expressed in the HF, which exhibited not only the co-expression pattern with the key factors to the HF development but also the activated expression in the August to October time point. Intriguingly, one of spatiotemporal lincRNAs, linc-chig1598 could be a potential regulator of distal-less homeobox 3 expression during the secondary hair follicle growth. Conclusions: This study will facilitate future studies aimed at unravelling the function of lincRNAs in hair follicle development. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are multi-potential cells that are able to proliferate and differentiate into other cell types. Much research has been done on the MSCs from the umbilical cord (UCMSCs) in human, mice, and avian, but little literature has been published about these cells in big livestock. Here, we choose Luxi cattle as the experimental animal, we describe an external culture of the UCMSCs from it and summarize the biological characteristics of these cells, e.g., morphologic appearance, surface antigens, colony-forming ability, gene expression, and differentiation potential were detected via using immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). The induced cells, osteoblast, lipoblast, hepatocyte, islet cells, and neurocyte were identified by Alizarin red staining, Oil-red-O staining, Periodic acid-schiff staining, and Dithizone staining and RT-PCR detection for specific genes. Results suggest that biological characteristics of the UCMSCs were similar to those of MSCs previously analyzed. The primary UCMSCs were sub-cultured to passage 32, the UCMSCs expressed gene CD29, CD44, CD73, CD90, and CD166, induced cells illustrated typical staining, and expressed specific genes, which indicate that the UCMSCs could be a novel alternative source of MSCs for experimental and clinical applications. [ABSTRACT FROM AUTHOR]

The hair follicle is a complex mini-organ in the skin that undergoes organ induction, morphogenesis, and regeneration. However, the accurate molecular mechanism of skin and hair diversity regulation is still elusive. The sheep is an animal model that can be used to further explore the mechanisms of skin and hair diversity. In this study, we carried out a transcriptomic analysis of the mRNA expression in the skin of Xinji fine-wool sheep at different growth stages (3 and 12 months old) and 12-month-old Tan sheep and explored the transcripts' relationship with hair follicle growth. A total of 1327 mRNAs and 67 transcription factors were identified to be differentially expressed in the different breeds and during different periods of skin development. The differentially expressed genes were enriched in GO terms represented by system development, multicellular organism development, animal organ development, and skin development, and three KEGG pathways typified those governing differences in skin structure. Combining protein–protein interaction networks of skin development (GO:0043588) and functional annotation, nine important candidate genes, namely, LAMA5, OVOL1, SRF, DHCR24, NGFR, SMO, CDSN, HOXC13, and KDF1, and many core genes with minor effects were confirmed to be associated with hair follicle development. Furthermore, members of the zf-C2H2 and homeobox transcription factor families, which were identified to play a crucial role in producing finer and denser wool, were mainly upregulated in 12-month-old Xinji fine-wool sheep when compared with expression in 12-month-old Tan sheep and 3-month-old Xinji fine-wool sheep. This study revealed the major–minor gene interactions in the developmental pathway and provided ideas for an in-depth understanding of the genetic structure and gene regulation in the skin/hair growth process. [ABSTRACT FROM AUTHOR]

[Display omitted] • We find two functional mutations driving the finer and denser wool production. • One C/A missense variant of KRT74 drive finer wool production in Chinese fine wool sheep. • The C-KRT74 can activate the KRT74 protein and affect wool fineness. • One T/C SNP in the regulatory region upstream of EDAR drive denser wool production in fine wool sheep. • The C-to-T mutation can upregulate EDAR mRNA expression and potentially affect wool density. Fine-wool sheep are the most common breed used by the wool industry worldwide. Fine-wool sheep have over a three-fold higher follicle density and a 50% smaller fiber diameter than coarse-wool sheep. This study aims to clarify the underlying genetic basis for the denser and finer wool phenotype in fine-wool breeds. Whole-genome sequences of 140 samples, Ovine HD630K SNP array data of 385 samples, including fine, semi-fine, and coarse wool sheep, as well as skin transcriptomes of nine samples were integrated for genomic selection signature analysis. Two loci at keratin 74 (KRT74) and ectodysplasin receptor (EDAR) were revealed. Fine-scale analysis in 250 fine/semi-fine and 198 coarse wool sheep narrowed this association to one C/A missense variant of KRT74 (OAR3:133,486,008, P = 1.02E-67) and one T/C SNP in the regulatory region upstream of EDAR (OAR3:61,927,840, P = 2.50E-43). Cellular over-expression and ovine skin section staining assays confirmed that C-KRT74 activated the KRT74 protein and specifically enlarged cell size at the Huxley's layer of the inner root sheath (P < 0.01). This structure enhancement shapes the growing hair shaft into the finer wool than the wild type. Luciferase assays validated that the C-to-T mutation upregulated EDAR mRNA expression via a newly created SOX2 binding site and potentially led to the formation of more hair placodes. Two functional mutations driving finer and denser wool production were characterized and offered new targets for genetic breeding during wool sheep selection. This study not only provides a theoretical basis for future selection of fine wool sheep breeds but also contributes to improving the value of wool commodities. [ABSTRACT FROM AUTHOR]

Skeletal muscle satellite cells (SMSCs) play an important role in regulating muscle growth and regeneration. Chromatin accessibility allows physical interactions that synergistically regulate gene expression through enhancers, promoters, insulators, and chromatin binding factors. However, the chromatin accessibility altas and its regulatory role in ovine myoblast differentiation is still unclear. Therefore, ATAC-seq and RNA-seq analysis were performed on ovine SMSCs at the proliferation stage (SCG) and differentiation stage (SCD). 17,460 DARs (differential accessibility regions) and 3732 DEGs (differentially expressed genes) were identified. Based on joint analysis of ATAC-seq and RNA-seq, we revealed that PI3K-Akt, TGF-β and other signaling pathways regulated SMSCs differentiation. We identified two novel candidate genes, FZD5 and MAP2K6 , which may affect the proliferation and differentiation of SMSCs. Our data identify potential cis regulatory elements of ovine SMSCs. This study can provide a reference for exploring the mechanisms of the differentiation and regeneration of SMSCs in the future. • ATAC-seq and RNA-seq analysis were performed on ovine SMSCs at the proliferation stage (SCG) and differentiation stage (SCD). • 17,460 DARs (differential accessibility regions) and 3732 DEGs (differentially expressed genes) were identified. • Based on joint analysis of ATAC-seq and RNA-seq, PI3K-Akt, TGF-βand other signaling pathways regulated SMSCs differentiation. • We identified two novel candidate genes, FZD5 and MAP2K6 ，which may affect the proliferation and differentiation of SMSCs. [ABSTRACT FROM AUTHOR]

Adipose-derived stem cells (ADSCs) that are enriched in adipose tissue with multilineage differentiation potential have become an important tool in therapeutic research and tissue engineering. Certain breeds of sheep exhibit a unique fat tail trait such that tail tissue accounts for approximately 10 % of body weight and can provide an excellent source of ADSCs. Here, we describe isolation of primary ADSCs from ovine embryonic fat tail tissues that displayed high self-renewal capacity, multilineage differentiation and excellent adipogenic ability. Through transcriptome analysis covering ADSCs differentiating into adipocytes, 37 transcription factors were involved in early transcriptional events that initiate a regulatory cascade of adipogenesis; the entire adipogenic activity consists of a reduction in proliferation ability and upregulation of genes related to lipid generation and energy metabolism, as well as several genes associated with myogenesis. Furthermore, Comparative transcriptome analysis across species (sheep, human, and mouse) revealed enhanced basal metabolic ability in differentiating ovine ADSCs, which may relate to the excellent adipogenic capability of these cells. We also identified a small evolutionarily conserved gene set, consisting of 21 and 22 genes exhibiting increased and decreased expression, respectively. Almost half (20) of these genes have not previously been reported to regulate adipogenesis in mammals. In this study, we identified important regulators that trigger ovine adipocyte differentiation, main biological pathways involved in adipogenesis as well as the evolutionarily conserved genes governing adipogenic process across species. Our study provides a novel excellent biomaterial and novel genes regulating adipogenesis for cellular transplantation therapy and investigations of fat metabolism. Transcriptomic characteristics of differentiating human, mouse, and sheep adipose-derived stem cells toward adipocyte, including the crucial genes encoding transcription factors with increased expression or decreased expression at each stage of adipogenesis of ovine ADSCs (inner circle), the specifically expressed genes by sheep (top arc) and human cells (bottom arc), as well as the evolutionary conservative genes set regulating adipogenesis in vitro across species. [Display omitted] • The clear physical distinction of fat tail, the large volume of fat tissue, and readily available donor animals makes fat tail sheep excellent low-cost sources of adipose-derived stem cells (ADSCs) for research and clinical applications. • We depicted the standard procedures to isolate ADSCs from ovine embryonic tail tissue and perform multi-lineages differentiation, and the isolated ADSCs exhibit excellent adipogenic ability. • Several important genes encoding transcription factors were involved in early transcriptional events that initiate a regulatory cascade of adipogenesis. • The enhanced basal metabolic ability in differentiating ovine ADSCs and one small evolutionarily conserved gene set were discovered by comparative transcriptome analysis across species. • In summary, this study provides a novel excellent biomaterial and novel genes regulating adipogenesis for cellular transplantation therapy and investigations of fat metabolism. [ABSTRACT FROM AUTHOR]

In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×106 pfu/mL and 1.59×1010 pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future. [ABSTRACT FROM AUTHOR]