ABSTRACT
BACKGROUND: Genome-wide association studies (GWAS) are an effective way to explore genotype-phenotype associations in humans, animals, and plants. Various GWAS methods have been developed based on different genetic or statistical assumptions. However, no single method is optimal for all traits and, for many traits, the putative single nucleotide polymorphisms (SNPs) that are detected by the different methods do not entirely overlap due to the diversity of the genetic architecture of complex traits. Therefore, multi-tool-based GWAS strategies that combine different methods have been increasingly employed. To take this one step further, we propose an ensemble-like GWAS strategy (E-GWAS) that statistically integrates GWAS results from different single GWAS methods. RESULTS: E-GWAS was compared with various single GWAS methods using simulated phenotype traits with different genetic architectures. E-GWAS performed stably across traits with different genetic architectures and effectively controlled the number of false positive genetic variants detected without decreasing the number of true positive variants. In addition, its performance could be further improved by using a bin-merged strategy and the addition of more distinct single GWAS methods. Our results show that the numbers of true and false positive SNPs detected by the E-GWAS strategy slightly increased and decreased, respectively, with increasing bin size and when the number and the diversity of individual GWAS methods that were integrated in E-GWAS increased, the latter being more effective than the bin-merged strategy. The E-GWAS strategy was also applied to a real dataset to study backfat thickness in a pig population, and 10 candidate genes related to this trait and expressed in adipose-associated tissues were identified. CONCLUSIONS: Using both simulated and real datasets, we show that E-GWAS is a reliable and robust strategy that effectively integrates the GWAS results of different methods and reduces the number of false positive SNPs without decreasing that of true positive SNPs.
Subject(s)
Genome-Wide Association Study , Polymorphism, Single Nucleotide , Humans , Animals , Swine , Genome-Wide Association Study/methods , Genetic Association Studies , PhenotypeABSTRACT
Since metastasis remains the primary reason for colorectal cancer (CRC) associated death, a better understanding of the molecular mechanism underlying CRC metastasis is urgently needed. Here, we elucidated the role of Cathepsin C (CTSC) in promoting CRC metastasis. The expression of CTSC was detected by real-time PCR and immunohistochemistry in the human CRC cohort. The metastatic capacities of CTSC-mediated metastasis were analyzed by in vivo metastasis model. Elevated CSTC expression was positively associated with tumor differentiation, tumor invasion, lymph node metastasis, and AJCC stage and indicated poor prognosis in human CRC. CTSC overexpression in CRC cells promoted myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) recruitment by the CSF1/CSF1R axis. In contrast, the knockdown of CSF1 reduced CTSC-mediated MDSCs and TAMs infiltration and CRC metastasis. Depletion of either MDSCs or TAMs decreased CTSC-mediated CRC metastasis. In human CRC tissues, CTSC expression was positively associated with intratumoral MDSCs and TAMs infiltration. Furthermore, the combination of CTSC inhibitor AZD7986 and anti-PD-L1 antibody blocked CTSC-induced CRC metastasis. CTSC overexpression promoted MDSCs and TAMs infiltration by CSF1/CSF1R axis. Interruption of this oncogenic loop may provide a promising treatment strategy for inhibiting CTSC-driven CRC metastasis.
Subject(s)
Cathepsin C , Colorectal Neoplasms , Humans , Cell Differentiation , Cell Line, Tumor , Colorectal Neoplasms/pathology , Lymphatic Metastasis , Neoplasm MetastasisABSTRACT
Ammonia toxicity to respiratory system in pig faming is of particular concern, but the molecular mechanism remains still unclear. The present study was devoted to assess the impacts of the ammonia exposure on the lung tissues based on a pig study using 80 ppm ammonia exposing to piglets for different days. The histology analysis revealed ammonia exposure induced lung injury and inflammatory response, as indicated by epithelial-mesenchymal transition (EMT), significant thickening of alveolar septa, infiltration of inflammatory cells and excessive mucus production. The transcriptome analysis revealed many more up-regulated genes in exposure groups when compared with the control group, and these genes were significantly enriched in the GO term of extracellular exosome, proteolysis, and regulation of circadian rhythm. The study discovered the induction of seven genes (CRY2, CIART, CREM, NR1D1, NR1D2, PER1 and PER3) that encode repressors of circadian clock. One gene (ARNTL) that encodes activator of circadian clock was down-regulated after ammonia exposure. The results of this study suggest that ammonia exposure disturbed the pulmonary circadian clock gene expression, which may establish new evidence for further understanding the toxicity of ammonia to lungs.
Subject(s)
Ammonia/toxicity , Circadian Clocks/drug effects , Lung Injury , Ammonia/metabolism , Animals , Circadian Clocks/genetics , Circadian Rhythm/drug effects , Circadian Rhythm/genetics , Gene Expression Profiling , Gene Regulatory Networks , Lung/metabolism , Swine , Transcription Factors/genetics , Up-RegulationABSTRACT
Porcine satellite cells (PSCs) play a vital role in the construction, development and self-renewal of skeletal muscle. In this study, PSCs were exposed to poly(I:C) stimulation to mimic viral infection during the proliferation and differentiation phases at 0, 12, 24 and 48â¯hours (h) of the stimulation. The untreated and treated PSCs were analyzed by the RNA-Seq technology. There were 88, 119, 104 and 95 genes being differentially expressed in 0â¯h vs 12â¯h treated, 12â¯h vs 24â¯h treated, 0â¯h vs 24â¯h treated and 24â¯h vs 48â¯h untreated comparison libraries, respectively. The GO terms analysis results showed that during the proliferation phase of treated PSCs, the up-regulated genes related to the immune system were highly expressed. In addition, the gene expressions associated with muscle structure development in response to growth factor emerged during the differentiation phase of untreated PSCs. The biological pathways associated with Influenza A, Toll-like receptor and chemokine signaling were revealed in PSCs following poly(I:C) stimulation. The differentially expressed genes were confirmed by quantitative real-time PCR. These findings expanded our understanding of gene expressions and signaling pathways about the infiltrated mechanism of the virus into PSCs.
Subject(s)
Cell Differentiation/genetics , Cell Proliferation/genetics , Muscle, Skeletal/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Gene Expression Profiling , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Gene Ontology , MicroRNAs/genetics , Microarray Analysis , Muscle, Skeletal/drug effects , Poly I-C/pharmacology , Swine , Toll-Like Receptors/geneticsABSTRACT
MicroRNA (miRNA) is a class of short non-coding RNA, which is about 22 bp in length. In mammals, miRNA exerts its funtion through binding with the 3°-UTR region of target genes and inhibiting their translation. Skeletal muscle development is a complex event, including: proliferation, migration and differentiation of skeletal muscle stem cells; proliferation, differentiation and fusion of myocytes; as well as hypertrophy, energy metabolism and conversion of muscle fiber types. The miRNA plays important roles in all processes of skeletal muscle development through targeting the key factors of different stages. Herein we summarize the miRNA related to muscle development, providing a better understanding of the skeletal muscle development.
Subject(s)
MicroRNAs/physiology , Muscle Development , Muscle, Skeletal/growth & development , Animals , Cell Differentiation , Cell Proliferation , Energy Metabolism , HumansABSTRACT
Circular RNAs (circRNAs) are a novel type of endogenous RNAs featuring stable structure and high tissue-specific expression. Recently, accumulating evidence has revealed that aberrant circRNAs expression plays important roles in carcinogenesis and tumor progression. However, the expression pattern and biological function of circRNAs in non small cell lung cancer (NSCLC) remain largely unknown. Therefore, the purpose of this study was to clarify the possible role of one of typical circRNAs, circRNA_100876 in NSCLC and to define its prognostic value in NSCLC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circRNA_100876 in tumor tissues and their adjacent nontumorous tissues in 101 patients with NSCLC. We found that the expression level of circRNA_100876 was significantly elevated in NSCLC tissues when compared with their adjacent nontumorous tissues (P=0.000). Moreover, there was a close correlation between the circRNA_100876 up-regulation expression and lymph node metastasis (P=0.001) and tumor staging (P=0.001) in NSCLC. In addition, Kaplan-Meier survival analysis demonstrated that the overall survival time of NSCLC patients with high circRNA_100876 expression was significantly shorter than those patients with low circRNA_100876 expression (P=0.000). In conclusion, our findings indicate that circRNA_100876 is closely related to the carcinogenesis of NSCLC and it might be served as a potential prognostic biomarker and therapeutic target for NSCLC.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/diagnosis , Lung Neoplasms/diagnosis , RNA/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , Prognosis , RNA, Circular , Up-RegulationABSTRACT
MicroRNAs (miRNAs) play important roles in the development of skeletal muscle. In our previous study, expression of miR-195 and miR-497 were shown to be upregulated during muscle development in pigs. In this study, we investigated the roles of these two miRNAs in myogenesis and analyzed their transcriptional regulation. Our results showed that miR-195 and miR-497 were upregulated during muscle development and myoblast differentiation. Moreover, miR-195 and miR-497 inhibited proliferation but not differentiation in C2C12 cells. Further investigation revealed that Igf1r, Insr, Ccnd2 and Ccne1 were directly targeted by miR-195 and miR-497 in myoblasts. In addition, we confirmed that miR-195 and miR-497, which shared the similar expression profiling, were negatively regulated by nuclear factor κB (NF-κB) in both myoblasts and skeletal muscle tissue. Our data illustrate that the signaling pathway NF-κB-miR-195/497-Igf1r/Insr-Ccnd2/Ccne1 plays important roles in myogenesis. Our study provides novel evidence for the roles of miR-195 and miR-497 in muscle development.
Subject(s)
Cyclin D2/genetics , Cyclin E/genetics , MicroRNAs/metabolism , Myoblasts/cytology , NF-kappa B/metabolism , Receptor, IGF Type 1/genetics , Receptor, Insulin/genetics , Animals , Base Sequence , Binding Sites , Cell Differentiation , Cell Line , Cell Proliferation , Cyclin D2/metabolism , Cyclin E/metabolism , Mice, Inbred BALB C , MicroRNAs/genetics , Molecular Sequence Data , Muscle Development , Myoblasts/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Receptor, IGF Type 1/metabolism , Receptor, Insulin/metabolism , Up-RegulationABSTRACT
The third generation of CRISPR/Cas9-mediated genome editing technology has been successfully applied to genome modification of various species including animals, plants and microorganisms. How to improve the efficiency of CRISPR/Cas9 genome editing and reduce its off-target effects has been extensively explored in this field. Using sgRNA (Small guide RNA) with high efficiency and specificity is one of the critical factors for successful genome editing. Several software have been developed for sgRNA design and/or off-target evaluation, which have advantages and disadvantages respectively. In this review, we summarize characters of 16 kinds online and standalone software for sgRNA design and/or off-target evaluation and conduct a comparative analysis of these different kinds of software through developing 38 evaluation indexes. We also summarize 11 experimental approaches for testing genome editing efficiency and off-target effects as well as how to screen highly efficient and specific sgRNA.
Subject(s)
CRISPR-Cas Systems/genetics , Genome/genetics , RNA Editing , RNA, Guide, Kinetoplastida/genetics , Humans , SoftwareABSTRACT
The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was evaluated according to mismatched number and "seed sequence" specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The software can be downloaded from BiooTools website (http://www.biootools.com/).
Subject(s)
CRISPR-Cas Systems , Endonucleases/metabolism , RNA, Guide, Kinetoplastida/metabolism , Software , Animals , Chickens , Computational Biology/methods , Humans , Internet , Mice , MicroRNAs/genetics , Programming Languages , RNA Precursors/genetics , Rats , Reproducibility of Results , Swine , User-Computer InterfaceABSTRACT
BACKGROUND: Litter size in pigs is a major factor affecting the profitability in the pig industry. The peri-implantation window in pigs is characterized by the coordinated interactions between the maternal uterine endometrium and the rapidly elongating conceptuses and represents a period of time during which a large percentage of the developing conceptuses are lost. However, the gene expression and regulatory networks in the endometrium contributing to the establishment of the maternal: placental interface remain poorly understood. RESULTS: We characterized the endometrial gene expression profile during the peri-implantation stage of development by comparing two breeds that demonstrate very different reproductive efficiencies. We employed the porcine Affymetrix GeneChip® to assay the transcriptomic profiles of genes expressed in the uterine endometrium obtained from Meishan and Yorkshire gilts (n = 4 for each breed) on day 12 of gestation (M12 and Y12, respectively). Total of 17,076 probesets were identified as "present" in at least two arrays. A mixed model-based statistical analysis predicted a total of 2,656 (q < 0.1) transcripts as differentially expressed between Meishan and Yorkshire pigs. Eighteen differentially expressed transcripts of interest were validated by quantitative real-time PCR. Gene ontology (GO) annotation revealed that the known functions of the differentially expressed genes were involved in a series of important biological processes relevant to early pregnancy establishment in the pig. CONCLUSIONS: The results identified endometrial gene expression profiles of two breeds differing in litter size and identified candidate genes that are related to known physiological pathways related to reproductive prolificacy. These findings provide a deeper understanding of molecular pathways differing between two breeds at the critical peri-implantation stage of pregnancy, which can be utilized to better understand the events contributing to pregnancy establishment in the pig.
Subject(s)
Endometrium/metabolism , Gene Expression Profiling , Animals , Cluster Analysis , Computational Biology , Female , Gene Expression Regulation , Gene Regulatory Networks , Litter Size , Molecular Sequence Annotation , Pregnancy , Reproducibility of Results , Swine , TranscriptomeABSTRACT
Anal atresia (i.e., anorectal malformations) is a severe disorder that occurs during the development of the distal hindgut in infants, swine, and many other mammals and has an unclear genetic background. Recently, the Shh-responsive transcription factor GLI2 has been shown as essential to the normal development of the hindgut, and QTL studies in pigs revealed that this gene may be an important candidate for anal atresia (AA). We used the pig as the model to study the contribution of GLI2 to AA. We revealed the genomic structure of the porcine GLI2 gene with 14 exons and obtained the porcine GLI2 mRNA sequence with a 4,656-bp ORF coding a 1,551-amino acid protein. We further scanned the genome-wide mutations in this gene by direct sequencing using three genomic DNA pools from the AA pigs, full-sibs of AA pigs, and unaffected pigs, respectively. Finally, 30 single nucleotide polymorphisms (SNPs) and one intronic 9-nucleotide (nt) deletion were identified. Of these SNPs, 23 are intronic, 6 are synonymous, and 1 (446 G>A) in exon 8 is nonsynonymous (365Met >Ile). NCOI-RFLP of the 446 G>A polymorphism suggested that the predominant genotypes were all GG and AG in the three pig groups. In addition, there was no significant difference among the three groups in allele frequencies, which demonstrated that this locus was not associated with AA in pigs. However, the 12 SNPs encompassing exon 4 to exon 8 showed strong linkage disequilibrium in the AA pigs, which indicated that the mutations somewhere in this region may contribute to AA in pigs. Therefore, further investigation in this region is needed to elucidate the underlying mutations involved in the porcine AA.
Subject(s)
Anus, Imperforate/genetics , Genome-Wide Association Study , Kruppel-Like Transcription Factors/genetics , Mutation , Swine/genetics , Animals , Anorectal Malformations , Anus, Imperforate/metabolism , Cloning, Molecular , Disease Models, Animal , Exons , Humans , Linkage Disequilibrium , Molecular Sequence Data , Polymorphism, Single Nucleotide , Zinc Finger Protein Gli2ABSTRACT
BACKGROUND: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. RESULTS: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome. CONCLUSIONS: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.
Subject(s)
Genomics , Immunity/genetics , Molecular Sequence Annotation , Swine/genetics , Swine/immunology , Animals , Cattle , Evolution, Molecular , Gene Duplication , Humans , Immunoglobulins/genetics , Mice , Models, Molecular , Protein Conformation , Receptors, Antigen, T-Cell/genetics , Receptors, KIR/genetics , Selection, Genetic , Species SpecificityABSTRACT
BACKGROUND: Network biology (systems biology) approaches are useful tools for elucidating the host infection processes that often accompany complex immune networks. Although many studies have recently focused on Haemophilus parasuis, a model of Gram-negative bacterium, little attention has been paid to the host's immune response to infection. In this article, we use network biology to investigate infection with Haemophilus parasuis in an in vivo pig model. RESULTS: By targeting the spleen immunogenome, we established an expression signature indicative of H. parasuis infection using a PCA/GSEA combined method. We reconstructed the immune network and estimated the network topology parameters that characterize the immunogene expressions in response to H. parasuis infection. The results showed that the immune network of H. parasuis infection is compartmentalized (not globally linked). Statistical analysis revealed that the reconstructed network is scale-free but not small-world. Based on the quantitative topological prioritization, we inferred that the C1R-centered clique might play a vital role in responding to H. parasuis infection. CONCLUSIONS: Here, we provide the first report of reconstruction of the immune network in H. parasuis-infected porcine spleen. The distinguishing feature of our work is the focus on utilizing the immunogenome for a network biology-oriented analysis. Our findings complement and extend the frontiers of knowledge of host infection biology for H. parasuis and also provide a new clue for systems infection biology of Gram-negative bacilli in mammals.
Subject(s)
Haemophilus Infections/genetics , Haemophilus parasuis/immunology , Immunity, Innate , Swine Diseases/immunology , Swine/immunology , Animals , Haemophilus Infections/veterinary , Haemophilus parasuis/pathogenicity , Humans , Spleen/immunology , Swine/microbiology , Swine Diseases/pathology , Systems BiologyABSTRACT
In November 2009, scientists from the US, UK, and other countries announced the complete genome sequence draft of the domestic pig. With the release of improved versions of the pig genome assembly and the increase of correctly assembled sequenced fragments over the past two years, it is particularly urgent to have the pig genes annotated at whole-genome level. This article is aimed at introducing an excellent manual annotation tool, Otterlace software, developed by Sanger institute. We used CFL1 (Cofilin 1) gene as an example to expound the usage of the three main components of Otterlace, Zmap, Blixem, and Dotter tools, and developed a practical procedure for manual annotations. We have analyzed 243 immune-related genes, among which 180 genes have been completely or partially annotated, offering novel information to the porcine functional genomics.
Subject(s)
Genome , Software , Swine/genetics , Animals , Base Sequence , Molecular Sequence DataABSTRACT
Signal transducers and activators of transcription (STATs) are members of a recently identified family of transcription factors that activate gene transcription in response to a number of different cytokines. STAT4 and STAT6 were activated by interleukin (IL)-12 and IL-4 stimulation, which were important for the generation of Th1 and Th2 cells. In this study, we cloned the cDNA sequences and analyzed the genomic structure of porcine STAT4 (poSTAT4) and STAT6 (poSTAT6) genes. Chromosome localization assigned these two genes to SSC15 and SSC5, and they were most closely linked to maker SWR1002 and DK. The RT-PCR revealed that both genes were expressed in eight diverse tissues, with the highest level in small intestine, followed by lung, kidney, muscle and stomach, whereas expressions in heart, liver and spleen were relatively weak. Transient transfection indicated that poSTAT4 and poSTAT6 proteins distributed throughout the whole porcine hip artery endothelial cell. A single nucleotide polymorphism (A/G), which can be recognized by restriction enzyme TaiI, was identified at the 3' untranslated region of poSTAT6, and genotyping results showed apparent variation in allele frequency between Chinese indigenous and western breeds.
Subject(s)
STAT4 Transcription Factor/genetics , STAT6 Transcription Factor/genetics , Swine/genetics , 3' Untranslated Regions , Animals , Cell Nucleus/metabolism , Chromosome Mapping , Gene Frequency , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Haplotypes , Humans , Mice , Organ Specificity , Polymorphism, Single Nucleotide , Protein Transport , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , STAT4 Transcription Factor/metabolism , STAT6 Transcription Factor/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
MicroRNAs (miRNAs) are small non-coding RNAs that participate in diverse biological processes including skeletal muscle development. MiR-214 is an miRNA that is differentially expressed in porcine embryonic muscle and adult skeletal muscle, suggesting that miR-214 may be related to embryonic myogenesis. In this study, the myoblast cell line C2C12 was used for functional analysis of miR-214 in vitro. The results showed that miR-214 was expressed both in myoblasts and in myotubes and was upregulated during differentiation. After treatment with an miR-214 inhibitor and culturing in differentiation medium, myoblast differentiation was repressed, as indicated by the significant downregulation of expression of the myogenic markers myogenin and myosin heavy chain (MyHC). Interestingly, myoblast proliferation was also repressed when cells were transfected with an miR-214 inhibitor and cultured in growth medium by real-time proliferation assay and cell cycle analysis. Our results showed that miR-214 regulates both proliferation and differentiation of myoblasts depending on the conditions.
Subject(s)
MicroRNAs/physiology , Myoblasts/cytology , Animals , Cell Cycle/genetics , Cell Differentiation/genetics , Cell Line , Cell Proliferation , Culture Media , Mice , Mice, Inbred C3H , Muscle Development/physiology , Muscle Fibers, Skeletal/physiology , Myoblasts/physiology , Myogenin/metabolism , Myosin Heavy Chains/metabolism , Oligonucleotides, Antisense/pharmacology , Up-RegulationABSTRACT
Due to specific recognization of DNA sequences and designability, zinc finger nucleases (ZFN) has been used in knock in and knock out genes. Because of high ratio of homologous recombination of DSB-GT induces by ZFN, ZFN technology has been considered as the most powerful tool for gene modification. It has been successfully used at cell or embryo levels in plants and animals. Under the rapid development of high affinity zinc finger protein (ZFP), this technique will be applied to genetic engineering and breeding extensively in the future. This review discussed the DNA recognization mechanism and double strand break gene targeting (DSB-GT) of zinc finger nucleases (ZFN). Some application examples of ZFN were summarized.
Subject(s)
Endonucleases/chemistry , Endonucleases/metabolism , Gene Targeting , Genetic Engineering , Zinc Fingers , Amino Acid Sequence , Animals , Endonucleases/genetics , Gene Targeting/instrumentation , Genetic Engineering/instrumentation , Humans , Molecular Sequence Data , Protein Binding , Protein ConformationABSTRACT
Caveolin-1 (Cav1) plays a critical role in the invasion of pathogenic microbes into host cells, yet little is known about porcine Cav1. In this study, we provide the molecular characterization of Cav1 in pigs following stimulation with LPS/polyinosinic-polycytidylic acid as well as during infection with Haemophilus parasuis. The porcine Cav1 gene is 35 kb long and is located at SSC18q21; two isoforms (Cav1-α and Cav1-ß) are produced by alternative splicing. Three point mutations were identified in the coding region of the gene, two of which were significantly associated with nine immunological parameters in Landrace pigs, including the Ab response against porcine reproductive and respiratory syndrome virus and lymphocyte counts. Promoter analysis indicated that NF-κB activates both Cav1 transcripts, but the forkhead gene family specifically regulates Cav1-ß in the pig. Porcine Cav1 is expressed ubiquitously, with Cav1-α more abundantly expressed than Cav1-ß in all tissues investigated. Basal expression levels of Cav1 in PBMCs are relatively similar across different pig breeds. LPS and polyinosinic-polycytidylic acid markedly induced the expression of Cav1 in porcine kidney-15 cells in vitro, likely through NF-κB activation. Pigs infected with H. parasuis exhibited decreased expression of Cav1, particularly in seriously impaired organs such as the brain. This study provides new evidence that supports the use of Cav1 as a potential diagnostic and genetic marker for disease resistance in animal breeding. In addition, our results suggest that Cav1 may be implicated in the pathogenesis of Glasser's disease, which is caused by H. parasuis.
Subject(s)
Caveolin 1/chemistry , Haemophilus Infections/immunology , Haemophilus Infections/metabolism , Haemophilus parasuis , Swine Diseases/immunology , Swine Diseases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cattle , Caveolin 1/biosynthesis , Caveolin 1/genetics , Caveolin 1/metabolism , Cell Line , Chickens , Down-Regulation/genetics , Down-Regulation/immunology , Female , Genetic Variation/immunology , Haemophilus Infections/genetics , Haemophilus parasuis/immunology , Humans , Lipopolysaccharides/pharmacology , Male , Mice , Molecular Sequence Data , Poly I-C/pharmacology , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , Rats , Species Specificity , Swine , Swine Diseases/genetics , ZebrafishABSTRACT
The domestic pig (Sus scrofa) is an important economic animal for meat production and as a suitable model organism for comparative genomics and biomedical studies. In an effort to gain further identification of miRNAs in the pig, we have applied the Illumina Solexa sequencing technology to carry out an in-depth analysis of the miRNA transcriptome in a pool of equal amounts of RNA from 16 different porcine tissues. From this data set, we identified 437 conserved and 86 candidate novel miRNA/miRNA* in the pig, corresponding to 329 miRNA genes. Compared with all the reported porcine miRNAs, the result showed that 112 conserved and 61 candidate novel porcine miRNA were first reported in this study. Further analysis revealed extensive sequence variations (isomiRs) of porcine miRNAs, including terminal isomiRs at both the 5' and 3' ends and nucleotide variants. Read counts of individual porcine miRNA spanned from a few reads to approximately 405541 reads, confirming the different level of expression of porcine miRNAs. Subsequently, the tissue expression patterns of 8 miRNAs were characterized by Northern blotting. The results showed that miR-145, miR-423-5p, miR-320, miR-26a, and miR-191 are ubiquitously expressed in diverse tissues, while miR-92, miR-200a, and miR-375 were selectively enriched and expressed in special tissues. Meanwhile, the expression of 8 novel porcine-specific miRNAs was validated by stem-loop RT-PCR, and one of these was detected by Northern blotting. Using the porcine miRNA array designed according to our Solexa results, 123 miRNAs were detected expression in porcine liver tissues. A total of 58 miRNAs showed differential expression between the Tongcheng (a Chinese indigenous fatty breed) and Large White pig breeds (a lean type pig). Taken together, our results add new information to existing data on porcine miRNAs and should be useful for investigating the biological functions of miRNAs in pig and other species.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , MicroRNAs/isolation & purification , Sus scrofa/genetics , Animals , Gene Expression , MicroRNAs/analysis , Organ Specificity , Species Specificity , SwineABSTRACT
The candidate gene approach is one of the most commonly used methods for identifying genes underlying disease traits. Advances in genomics have greatly contributed to the development of this approach in the past decade. More recently, with the explosion of genomic resources accessible via the public Web, digital candidate gene approach (DigiCGA) has emerged as a new development in this field. DigiCGA, an approach still in its infancy, has already achieved some primary success in cancer gene discovery. However, a detailed discussion concerning the applications of DigiCGA in cancer gene identification has not been addressed. This chapter will focus on discussing DigiCGA in a generalized sense and its applications to the identification of cancer genes, including the cancer gene resources, application status, platform and tools, challenges, and prospects.