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1.
Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily that functions as a negative regulator
of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function
of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5′ and 3′ flanking
sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage
domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart.
It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several
types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further,
it was demonstrated that the 5′ flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures. 相似文献
2.
CRISPR/Cas9‐mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass 下载免费PDF全文
X. Wang Y. Niu J. Zhou H. Zhu B. Ma H. Yu H. Yan J. Hua X. Huang L. Qu Y. Chen 《Animal genetics》2018,49(1):43-51
Genetic engineering in livestock has been greatly enhanced through the use of artificial programmed nucleases such as the recently emerged clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) system. We recently reported our successful application of the CRISPR/Cas9 system to engineer the goat genome through micro‐injection of Cas9 mRNA and sgRNAs targeting MSTN and FGF5 in goat embryos. The phenotypes induced by edited loss‐of‐function mutations of MSTN remain to be evaluated extensively. We demonstrate the utility of this approach by disrupting MSTN, resulting in enhanced body weight and larger muscle fiber size in Cas9‐mediated gene‐modified goats. The effects of genome modifications were further characterized by H&E staining, quantitative PCR, Western blotting and immunofluorescence staining. Morphological and genetic analyses indicated the occurrence of phenotypic and genotypic modifications. We further provide sufficient evidence, including breeding data, to demonstrate the transmission of the knockout alleles through the germline. By phenotypic and genotypic characterization, we demonstrated the merit of using the CRISPR/Cas9 approach for establishing genetically modified livestock with an enhanced production trait. 相似文献
3.
4.
Junjie Luo Zhiyuan Song Shengli Yu Dan Cui Benli Wang Fangrong Ding Song Li Yunping Dai Ning Li 《PloS one》2014,9(4)
Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle. 相似文献
5.
Haploinsufficiency of myostatin protects against aging‐related declines in muscle function and enhances the longevity of mice 下载免费PDF全文
Christopher L. Mendias Konstantin I. Bakhurin Jonathan P. Gumucio Mark V. Shallal‐Ayzin Carol S. Davis John A. Faulkner 《Aging cell》2015,14(4):704-706
The molecular mechanisms behind aging-related declines in muscle function are not well understood, but the growth factor myostatin (MSTN) appears to play an important role in this process. Additionally, epidemiological studies have identified a positive correlation between skeletal muscle mass and longevity. Given the role of myostatin in regulating muscle size, and the correlation between muscle mass and longevity, we tested the hypotheses that the deficiency of myostatin would protect oldest-old mice (28–30 months old) from an aging-related loss in muscle size and contractility, and would extend the maximum lifespan of mice. We found that MSTN+/− and MSTN−/− mice were protected from aging-related declines in muscle mass and contractility. While no differences were detected between MSTN+/+ and MSTN−/− mice, MSTN+/− mice had an approximately 15% increase in maximal lifespan. These results suggest that targeting myostatin may protect against aging-related changes in skeletal muscle and contribute to enhanced longevity. 相似文献
6.
Qiang Wang Cheng Ji Jinxiu Huang Feiyun Yang Haiyan Zhang Ling Liu Jingdong Yin 《Molecular biology reports》2011,38(1):319-325
Lipin1 has been documented to play an important role in adipogenesis. In the present study, the mRNA expression level of lipin1
and its isoforms in longissimus dorsi muscle were determined by semi-quantification RT-PCR in lean PIC and obese Rongchang pigs. Further, we determined mRNA expression
for lipin1 and its two isoforms in Rongchang obese pigs which had either a high or low intramuscular fat content. We demonstrate
for the first time that porcine lipin1 has two alternative forms, lipin-α and lipin-β. Unlike mice and humans where the lipin-β
has 99 more nucleotides than lipin-α, we found that in swine, lipin-β has 108 more nucleotides than lipin-α. Our results indicate
that the longissimus dorsi muscle of Rongchang obese pigs have a higher level of mRNA expression for lipin1 and its isoforms than PIC lean pigs. Furthermore,
Rongchang pigs with higher intramuscular fat content had a higher lipin1 and lipin-β mRNA expression in longissimus dorsisi muscle than Rongchang pigs with lower intramuscular fat content (P < 0.05), whereas no difference was seen in lipin-α mRNA expression between Rongchang pigs with high or low intramuscular
fat. The ratio of lipin-β mRNA to lipin-α mRNA was also significantly different between Rongchang pigs distinguished by a
high intramuscular fat content compared with those with low intramuscular fat (P < 0.05). These data suggested that the lipin1 gene may have a crucial effect on body lipid accumulation in pigs, whereas
the lipin-β isoform may play an important role in intramuscular fat deposition in obese pigs. 相似文献
7.
Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity 总被引:1,自引:0,他引:1
Stinckens A Luyten T Bijttebier J Van den Maagdenberg K Dieltiens D Janssens S De Smet S Georges M Buys N 《Animal genetics》2008,39(6):586-596
Myostatin (MSTN), a transforming growth factor beta superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5' and 3' UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks. 相似文献
8.
《Animal : an international journal of animal bioscience》2021,15(6):100220
Differences in producing performance and organoleptic meat characteristics among pig genotypes and/or producing types are widely known. These parameters are also subjected to the animal’s development, feeding and management. Detailed knowledge of the effects of production phase (PP), pig producing type (PT), dietary protein availability and their interactions on nutrient digestibility, nitrogen balance and protein metabolism is essential information to improve precision feeding techniques. The experiment was a 2 (PP) × 2 (PT) × 2 (diet) factorial design conducted with 32 male pigs, 16 entire F2 pigs progeny of Pietrain sires and Duroc × Landrace dams, and 16 castrated purebred Durocs belonging to two production phases (growing: 29.5 ± 3.19 v. fattening: 88.6 ± 6.26 kg BW), and assigned to one of two dietary CP levels, either standard (SP: 17% in growing and 15% in fattening) or low (LP: 15% in growing and 13% in fattening). Viscera and muscle fractional protein synthesis rates (FSRs; %/day) were conducted through a single infusion of 15% L-[ring-2H5]-phenylalanine, with subsequent blood sampling from 12 to 40 min, and sample collection of liver, duodenum, biceps femoris and longissimus dorsi skeletal muscles after sacrifice. Fattening animals acquired a greater feed ingestion capacity, average daily gain (P < 0.01) and apparent ileal digestibility, whereas growing pigs showed higher FSRs in both viscera (duodenum and liver) and in longissimus dorsi. F2 pigs showed higher average daily gain, nitrogen retention rates and FSR in liver and longissimus dorsi (P < 0.01). Nevertheless, apparent ileal digestibility in all essential amino acids was lower in F2 compared with Duroc pigs (P < 0.05). Protein metabolism was barely influenced by dietary CP content, although animals fed LP registered the lowest apparent ileal digestibility for CP and also for most of the essential amino acids compared with SP-fed pigs. This information may reveal differences in amino acid requirements between both PTs, with Duroc pigs receiving excess of dietary amino acids. 相似文献
9.
M. Crispo A. P. Mulet L. Tesson N. Barrera F. Cuadro P. C. dos Santos-Neto T. H. Nguyen A. Crénéguy L. Brusselle I. Anegón A. Menchaca 《PloS one》2015,10(8)
While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock. 相似文献
10.
Kankan Wang Xiaochun Tang Zicong Xie Xiaodong Zou Mengjing Li Hongming Yuan Nannan Guo Hongsheng Ouyang Huping Jiao Daxin Pang 《Transgenic research》2017,26(6):799-805
CRISPR/Cas9 has emerged as one of the most popular genome editing tools due to its simple design and high efficiency in multiple species. Myostatin (MSTN) negatively regulates skeletal muscle growth and mutations in myostatin cause double-muscled phenotype in various animals. Here, we generated myostatin mutation in Erhualian pigs using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. The protein level of myostatin precursor decreased dramatically in mutant cloned piglets. Unlike myostatin knockout Landrace, which often encountered health issues and died shortly after birth, Erhualian pigs harboring homozygous mutations were viable. Moreover, myostatin knockout Erhualian pigs exhibited partial double-muscled phenotype such as prominent muscular protrusion, wider back and hip compared with wild-type piglets. Genome editing in Chinese indigenous pig breeds thus holds great promise not only for improving growth performance, but also for protecting endangered genetic resources. 相似文献
11.
Anna Granlund Marianne Jensen-Waern Birgitta Essén-Gustavsson 《Acta veterinaria Scandinavica》2011,53(1):20
Background
AMP-activated protein kinase (AMPK) plays an important role in the regulation of glucose and lipid metabolism in skeletal muscle. Many pigs of Hampshire origin have a naturally occurring dominant mutation in the AMPK γ3 subunit. Pigs carrying this PRKAG3 (R225Q) mutation have, compared to non-carriers, higher muscle glycogen levels and increased oxidative capacity in m. longissimus dorsi, containing mainly type II glycolytic fibres. These metabolic changes resemble those seen when muscles adapt to an increased physical activity level. The aim was to stimulate AMPK by exercise training and study the influence of the PRKAG3 mutation on metabolic and fibre characteristics not only in m. longissimus dorsi, but also in other muscles with different functions. 相似文献12.
13.
《Archives of animal nutrition》2013,67(4):317-324
Abstract This experiment was carried out to study the influence of age at the beginning of the free-range fattening period (traditional pigs, TP, age 12 months vs. young pigs, YP, age 8 months) on the performance of Iberian pigs. During 152 days prior to the fattening period, TP and YP pigs received 1.7 and 2.6 kg feed per day, respectively. During fattening, TP pigs had a higher average daily gain (p < 0.05) than YP pigs. The proportions of PUFA and n-3 fatty acids of the outer and inner layers of subcutaneous backfat were higher in TP than in YP pigs (p < 0.05), while the proportions of C16:0 and SFA in the inner layer of subcutaneous backfat were greater in YP than in TP pigs (p < 0.05). The ratio of n-6/n-3 in subcutaneous backfat was lower in TP than in YP pigs (p < 0.05). The percentage of intramuscular fat in longissimus dorsi muscle was higher in TP than in YP pigs (p < 0.05). The relationship between the percentage of intramuscular fat in longissimus dorsi muscle and average daily gain during the free-range fattening period adjusted to a quadratic function (p < 0.05). The concentration of α- and γ-tocopherol in subcutaneous backfat at slaughter was significantly higher in TP than in YP pigs (p < 0.05). It is concluded that Iberian pigs that have 8 months of age at the beginning of free-range feeding have adequate commercial quality. 相似文献
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15.
Xiao-Xu Xing Mei-Fu Xuan Long Jin Qing Guo Zhao-Bo Luo Jun-Xia Wang Qi-Rong Luo Guang-Lei Zhang Cheng-Du Cui Zheng-Yun Cui Jin-Dan Kang Xi-Jun Yin 《Biotechnology letters》2017,39(12):1811-1819
Objectives
To explore the effects of heterozygous myostatin-knockout (MSNT+/?) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs.Results
The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN+/? pigs at birth than in wild-type (WT) pigs. MSTN+/? pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was ~ threefold higher in MSTN+/? pigs compared with WT pigs.Conclusion
MSTN+/? pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.16.
Secreted frizzled-related protein 3 (sFRP3), encoded by the gene FRZB, is a member of the sFRP family with important roles in inhibition of the Wnt signalling pathway through competitive binding of the Wnt receptor. Here, we investigated pig FRZB as a candidate gene for growth traits and identified three polymorphic sites, an insertion (A-532B) and two SNPs (G636A and C650T) in its 5′-UTR. The genotype distributions of G636A and C650T were significantly different among mini-type indigenous (Diannan Small-ear and Tibetan), normal indigenous (Laiwu and Huai), and introduced (Large Yorkshire and Landrace) breeds. In semi-quantitative PCR expression analysis, expression of FRZB mRNA was abundant in tissues of hypophysis, longissimus dorsi muscle, and adipose tissues, and low in the heart, hypothalamus, and brain. Quantitative determination of mRNA level and protein expression analysis were corresponding. The results demonstrated that FRZB gene expression in longissimus dorsi muscle and liver tissue was significantly higher in Diannan Small-ear and Tibetan pigs than in the Large Yorkshire breed (P < 0.05); however, in back fat tissue, the expression was significantly higher in Diannan Small-ear pig than in Tibetan or Large Yorkshire breeds (P < 0.05). Given the known growth and fat characteristics of the breeds, these results indicate that FRZB expression has a negative association with muscle growth and a positive association with fat deposition. In conclusion, FRZB may be a major candidate gene for growth traits in pigs. 相似文献
17.
Xiaoping Su Kuiqing Cui Shanshan Du Hongli Li Fenghua Lu Deshun Shi Qingyou Liu 《In vitro cellular & developmental biology. Animal》2018,54(5):375-383
Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutation efficiency in fibroblasts was as high as 87.5% in pig and 78.9% in buffalo. We then obtained single-cell clones with mutations at the specific sites of the GDF8 gene by screening with G418 in fibroblasts of pig and buffalo. In addition, the frequencies of Cas9/gRNA-mediated mutations were at 36 and 25% in the intracytoplasmic sperm injection embryos of pig and in vitro fertilization embryos of buffalo, respectively. Our work demonstrates that the Cas9/gRNA system is a highly efficient and fast tool for genome editing in cultured cells and embryos of Debao pig and swamp buffalo. These results can be helpful for the establishment of a new animal strain that can generate more meat. 相似文献
18.
Ladybird-like genes were recently identified in mammals. The first member characterized, Lbx1, is expressed in developing skeletal muscle and the nervous system. However, little is known about the porcine Lbx1 gene. In the present study, we cloned and characterized Lbx1 from porcine muscle. RT-PCR analyses showed that Lbx1 was highly expressed in porcine skeletal muscle tissues. And we provide the first evidence that Lbx1 has a certain regulated expression pattern during the postnatal period of the porcine skeletal muscle development. Lbx1 gene expressed at higher levels in biceps femoris muscles compared with masseter, semitendinosus and longissimus dorsi muscles in Meishan pigs. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover,
single nucleotide polymorphism (SNP) scanning in the Lbx1 genomic fragment identified two mutations, g.752A>G and g.−1559C>G. Association analysis in our experimental pig populations
showed that the mutation of g.752A>G was significantly associated with loin muscle area (P < 0.05) and internal fat rate (P < 0.05). Our results suggest that the Lbx1 gene might be a candidate gene of carcass traits and provide useful information for further studies on its roles in porcine
skeletal muscle. 相似文献
19.
Long Jin Zhi Jiang Yudong Xia Ping’er Lou Lei Chen Hongmei Wang Lu Bai Yanmei Xie Yihui Liu Wei Li Bangsheng Zhong Junfang Shen An’an Jiang Li Zhu Jinyong Wang Xuewei Li Mingzhou Li 《BMC genomics》2014,15(1)