Project description:Molecular cloning and bioinformatics analysis of annexin A2 (ANXA2) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially.The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer (Cervus Nippon hortulorum) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR).The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus. Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period).ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.
Project description:The antler regeneration has been well studied for the past two decades and adopted in the regenerative medicine model for studying on developmental biology. Despite our growing knowledge of functional molecules regulating antler regeneration, we still do not know whether antler from different deer species possess the exact same mechanism or not. Our previous comparative study between sika deer and red deer suggests that the metabolic pathways between them are profoundly different based on protein level. Therefore, the metabolomic technology is used to identify and quantify the metabolites in antler samples, providing interesting insights into differential metabolite profile of antlers between sika deer and red deer. The distinct metabolic characteristics of sika deer compared to red deer provide an opportunity to explain why the red deer antler with a larger size. The enrichment analysis of differential metabolites showed that three pathways including glycine and serine metabolism, methionine metabolism, and pterine biosynthesis had a significant difference between two antler groups.
Project description:Deer antler, as the only mammalian regenerative appendage, provides an optimal model to study regenerative medicine. Antler harvested from red deer or sika deer were mainly study objects used to disclose the mechanism underlying antler regeneration over past decades. A previous study used proteomic technology to reveal the signaling pathways of antler stem cell derived from red deer. Moreover, transcriptome of antler tip from sika deer provide us with the essential genes, which regulated antler development and regeneration. However, antler comparison between red deer and sika deer has not been well studied. In our current study, proteomics were employed to analyze the biological difference of antler regeneration between sika deer and red deer. The proteomics profile was completed by searching the UniProt database, and differentially expressed proteins were identified by bioinformatic software. Thirty-six proteins were highly expressed in red deer antler, while 144 proteins were abundant in sika deer. GO and KEGG analysis revealed that differentially expressed proteins participated in the regulation of several pathways including oxidative phosphorylation, ribosome, extracellular matrix interaction, and PI3K-Akt pathway.
Project description:BACKGROUND:Sika deer is one of the most popular and valued animals in China. However, few studies have been conducted on the microsatellite of Sika deer, which has hampered the progress of genetic selection breeding. To develop and characterize a set of microsatellites for Sika deer which provide helpful information for protection of Sika deer natural resources and effectively increase the yield and quantity of velvet antler. RESULTS:We conducted a transcriptome survey of Sika deer using next-generation sequencing technology. One hundred eighty-two thousand two hundred ninety-five microsatellite markers were identified in the transcriptome, 170 of 200 loci were successfully amplified across panels of 140 individuals from Shuangyang Sika deer population. And 29 loci were found to be obvious polymorphic. Number of alleles is from 3 to 14. The expected heterozygosity ranged from 0.3087 to 0.7644. The observed heterozygosity ranged from 0 to 0.7698. The polymorphism information content values of those microsatellites varied ranged from 0.2602 to 0.7507. The marker-trait association was tested for 6 important and kernel characteristics of two-branched velvet antler in Shuangyang Sika deer through one-way analysis of variance. The results showed that marker-trait associations were identified with 8 different markers, especially M009 and M027. CONCLUSIONS:This study not only provided a large scale of microsatellites which were valuable for future genetic mapping and trait association in Sika deer, but also offers available information for molecular breeding in Sika deer.
Project description:Background:Deer antlers have become a valuable model for biomedical research due to the capacities of regeneration and rapid growth. However, the molecular mechanism of rapid antler growth remains to be elucidated. The aim of the present study was to compare and explore the molecular control exerted by the main beam and brow tine during rapid antler growth. Methods:The main beams and brow tines of sika deer antlers were collected from Chinese sika deer (Cervus nippon) at the rapid growth stage. Comparative transcriptome analysis was conducted using RNA-Seq technology. Differential expression was assessed using the DEGseq package. Functional Gene Ontology (GO) enrichment analysis was accomplished using a rigorous algorithm according to the GO Term Finder tool, and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was accomplished with the R function phyper, followed by the hypergeometric test and Bonferroni correction. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to verify the RNA levels for differentially expressed mRNAs. Results:The expression levels of 16 differentially expressed genes (DEGs) involved in chondrogenesis and cartilage development were identified as significantly upregulated in the main beams, including transcription factor SOX-9 (Sox9), collagen alpha-1(II) chain (Col2a1), aggrecan core protein (Acan), etc. However, the expression levels of 17 DEGs involved in endochondral ossification and bone formation were identified as significantly upregulated in the brow tines, including collagen alpha-1(X) chain (Col10a1), osteopontin (Spp1) and bone sialoprotein 2 (Ibsp), etc. Conclusion:These results suggest that the antler main beam has stronger growth capacity involved in chondrogenesis and cartilage development compared to the brow tine during rapid antler growth, which is mainly achieved through regulation of Sox9 and its target genes, whereas the antler brow tine has stronger capacities of endochondral bone formation and resorption compared to the main beam during rapid antler growth, which is mainly achieved through the genes involved in regulating osteoblast and osteoclast activities. Thus, the current research has deeply expanded our understanding of the intrinsic molecular regulation displayed by the main beam and brow tine during rapid antler growth.
Project description:<h4>Background</h4>Deer antler is the only mammalian organ that can be completely regenerated every year. Its periodic regeneration is regulated by multiple factors, including transforming growth factor ? (TGF-?). This widely distributed multi-functional growth factor can control the proliferation and differentiation of many types of cell, and it may play a crucial regulatory role in antler regeneration. This study explored the role of TGF-?1 during the rapid growth of sika deer antler.<h4>Methods</h4>Three CRISPR-Cas9 knockout vectors targeting the TGF-?1 gene of sika deer were constructed and packaged with a lentiviral system. The expression level of TGF-?1 protein in the knockout cell line was determined using western blot, the proliferation and migration of cartilage cells in vitro were respectively determined using EdU and the cell scratch test, and the expression levels of TGF-? pathway-related genes were determined using a PCR array.<h4>Results</h4>Of the three gRNAs designed, pBOBI-gRNA2 had the best knockout effect. Knockout of TGF-?1 gene inhibits the proliferation of cartilage cells and enhances their migration in vitro. TGF-? signaling pathway-related genes undergo significant changes, so we speculate that when the TGF-? pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role.<h4>Conclusions</h4>TGF-?1 is a newly identified regulatory factor of rapid growth in sika deer antler.
Project description:Deer antler preparations have been used to strengthen bones for centuries. It is particularly rich in collagen type I. This study aimed to unravel part of the purported bioremedial effect of Sika deer antler collagen type I (SDA-Col I) on bone marrow mesenchymal stem cells. The results suggest that SDA-Col I might be used to promote and regulate osteoblast proliferation and differentiation. SDA-Col I might potentially provide the basis for novel therapeutic strategies in the treatment of bone injury and/or in scaffolds for bone replacement strategies. Finally, isolation of SDA-Col I from deer antler represents a renewable, green, and uncomplicated way to obtain a biomedically valuable therapeutic.
Project description:we used proteomic technology to disclose the difference of antler regeneration between red deer and sika deer. Through functional analysis, we obtained differentially expressed proteins and the pathway involved in antler regeneration between two groups
Project description:BACKGROUND:Deer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration. METHODS:To identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (>?60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies. RESULTS:Cell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6-11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10. CONCLUSION:Using a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.
2018-01-01 | S-EPMC6208050 | BioStudies
Project description:Transcriptome analysis of Sika deer antler at different stages