This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPβ, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPβ, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.
Animals ; MicroRNAs/metabolism* ; Goats/genetics* ; PPAR gamma/metabolism* ; Adipogenesis/genetics* ; Cell Differentiation/genetics* ; Luciferases ; RNA, Messenger

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.
Animals ; Goats/genetics* ; Cell Differentiation/genetics* ; Adipogenesis/genetics* ; Gene Expression Regulation ; Proteins/genetics* ; Cloning, Molecular

The purpose of this study was to clone and characterize the ZFP36L1 (zinc finger protein 36-like 1) gene, clarify its expression characteristics, and elucidate its expression patterns in different tissues of goats. Samples of 15 tissues from Jianzhou big-eared goats, including heart, liver, spleen, lung and kidney were collected. Goat ZFP36L1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR), then the gene and protein sequence were analyzed by online tools. Quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression level of ZFP36L1 in intramuscular preadipocytes in different tissues and adipocytes of goat at different differentiation stages. The results showed that the length of ZFR36L1 gene was 1 224 bp, and the coding sequence (CDS) region was 1 017 bp, encoding 338 amino acids, which was a non-secretory unstable protein mainly located in nucleus and cytoplasm. Tissue expression profile showed that ZFP36L1 gene was expressed in all selected tissues. In visceral tissues, the small intestine showed the highest expression level (P < 0.01). In muscle tissue, the highest expression level was presented in longissimus dorsi muscle (P < 0.01), whereas the expression level in subcutaneous adipose tissue was significantly higher than that in other tissues (P < 0.01). The results of induced differentiation showed that the expression of this gene was up-regulated during adipogenic differentiation of intramuscular precursor adipocytes (P < 0.01). These data may help to clarify the biological function of the ZFP36L1 gene in goat.
Animals ; Goats/genetics* ; Amino Acid Sequence ; Liver ; Cloning, Molecular

The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P < 0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P < 0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P < 0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P < 0.01) and a significant increase in FASN gene expression (P < 0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.
Animals ; Lipogenesis/genetics* ; Adipogenesis/genetics* ; Goats/genetics* ; Adipocytes ; Cell Differentiation/genetics* ; Sequence Analysis ; Cloning, Molecular

The aim of this study was to investigate the effect of activating transcription factor 3 (ATF3) on the differentiation of intramuscular preadipocytes in goat, and to elucidate its possible action pathway at the molecular level. In this study, the recombinant plasmid of goat pEGFP-N1-ATF3 was constructed, and the intramuscular preadipocytes were transfected with liposomes. The relative expression levels of adipocyte differentiation marker genes were detected by quantitative real-time PCR (qRT-PCR). After transfection of goat intramuscular preadipocytes with the goat pEGFP-N1-ATF3 overexpression vector, it was found that the accumulation of lipid droplets was inhibited, and the adipocyte differentiation markers PPARγ, C/EBPα and SREBP1 were extremely significantly down-regulated (P < 0.01), while C/EBPβ and AP2 were significantly down-regulated (P < 0.05). The ATF3 binding sites were predicted to exist in the promoter regions of PPARγ, C/EBPα and AP2 by the ALGGEN PROMO program. The overexpression of goat ATF3 inhibits the accumulation of lipid droplets in intramuscular preadipocytes, and this effect may be achieved by down-regulating PPARγ, C/EBPα and AP2. These results may facilitate elucidation of the regulatory mechanism of ATF3 in regulating the differentiation of goat intramuscular preadipocytes.
3T3-L1 Cells ; Activating Transcription Factor 3/pharmacology* ; Adipocytes ; Adipogenesis/genetics* ; Animals ; CCAAT-Enhancer-Binding Protein-alpha/pharmacology* ; Cell Differentiation ; Goats ; Mice ; PPAR gamma/metabolism*

In this study, we cloned the complete sequence coding for aminoacids in protein (CDS) of goat ST13 gene, analyzed the bioinformation of it, and explored the expression pattern in different goat tissues and goat subcutaneous preadipocytes at different differentiation stages. To be specific, ST13 gene was cloned by reverse transcription PCR (RT-PCR), and the bioinformation was analyzed by online tools or software. The expression in various goat tissues and subcutaneous preadipocytes at different differentiation stages was detected by quantitative reverse transcription PCR (qRT-PCR). The results showed that the cloned goat ST13 gene was 1 380 bp, with CDS of 1 101 bp, encoding 366 amino acids. Protein prediction results showed that ST13 had 26 phosphorylation sites and that some sequences were highly hydrophilic and unstable. Moreover, ST13 was a non-transmembrane and non-secretory protein. Subcellular localization demonstrated that ST13 was mostly distributed in the nucleus (69.6%). Phylogeny analysis suggested that goat ST13 had the highest identity to sheep ST13. Tissue expression pattern showed that ST13 gene expressed in all of the collected 13 tissues of goat, including heart, liver, spleen, lung and kidney, especially in triceps brachii and subcutaneous fat (P < 0.01) and that the expression among heart, liver, spleen, lung, kidney, large intestine, small intestine and pancreas was insignificantly different (P > 0.05). In addition, according to the temporal expression pattern in adipocytes, the expression of ST13 was up-regulated in differentiated adipocytes, and the expression was the highest at the 108th hour of induction, significantly higher than that at other time points (P < 0.01). In conclusion, this gene expresses in various tissues of goat and regulates the differentiation of goat subcutaneous adipocytes.
Adipocytes ; Animals ; Cloning, Molecular ; Goats/genetics* ; Liver ; Phylogeny ; Real-Time Polymerase Chain Reaction ; Sheep

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.
Animals ; Bone Morphogenetic Proteins ; genetics ; Chromosomes, Artificial, Bacterial ; DNA Transposable Elements ; Fibroblasts ; Goats ; genetics ; Keratins ; genetics ; Transfection

Anaplasma species are obligate intracellular pathogens that can cause tick-borne diseases in mammalian hosts. To date, very few studies of their occurrence in Korean native goats (Capra aegagrus hircus) have been reported. In the present study, we investigated Anaplasma infection of Korean native goats on Jeju Island, Republic of Korea, and performed phylogenetic analysis based on the 16S rRNA gene sequences. Our results showed that Anaplasma infection was found mostly in adult female goats. The phylogenetic tree revealed that the 7 sequences identified in Korean native goats could belong to Anaplasma sp. and were distinct from A. marginale, A. centrale, and A. ovis. The results indicated that the sequences identified to belong to Anaplasma were closely related to sequences isolated from goats in China and were clustered within the same group. To our knowledge, this is the first study to detect Anaplasma sp. infection in Korean native goats.
Anaplasma/classification/genetics/*isolation & purification ; Anaplasmosis/*microbiology ; Animals ; Female ; Goat Diseases/*microbiology ; Goats ; Islands ; Male ; Molecular Sequence Data ; Phylogeny ; Republic of Korea

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.
Amino Acid Sequence ; Animals ; Base Sequence ; Cats ; *Genetic Variation ; Goats ; Humans ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/chemistry/*genetics/metabolism ; Sequence Alignment ; Sheep ; Superoxide Dismutase/chemistry/*genetics/metabolism ; Toxoplasma/classification/*enzymology/genetics/isolation & purification ; Toxoplasmosis/*parasitology ; Toxoplasmosis, Animal/*parasitology