• Asian-Australasian Journal of Animal Sciences
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• v.30 no.5
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• pp.743-748
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• 2017

Objective: Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells. Methods: Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. Results: Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. Conclusion: The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.4
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• pp.224-229
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• 2007

Muscle tissue expresses many muscle-specific genes, including myostatin (also known as GDF8) that is a member of the transforming growth factor-beta superfamily. Myostatin (MSTN) negatively regulates mammalian skeletal muscle growth and development by inhibiting myoblast proliferation. Mice and cattle possessing mutant MSTN alleles display a 'double muscling' phenotype characterized by extreme skeletal muscle hypertrophy and/or hyperplasia. In this study, we first have characterized partial cDNA of a MSTN gene from the muscle tissue in the F. chinensis and examined its expression pattern in various tissues. The partial MSTN gene (GenBank accession number EU 131093) in the F. chinensis was 1134 bp, encoding for 377 amino acids that showed 63-93% amino acid similarity to other vertebrate MSTNs, containing a conserved proteolytic cleavage site (RXRR) and conserved cysteine residues in the C-terminus. Based on a RT-PCR, the MSTN gene was expressed in the all tissues of F. chinensis used in this study.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.10
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• pp.1500-1507
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• 2016

Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause "double-muscling" trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

• Proceedings of the Korean Aquaculture Society Conference
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• 2006.05a
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• pp.351-352
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• 2006

• Proceedings of the Korea Society of Poultry Science Conference
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• 1999.11a
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• pp.62-63
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• 1999

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.10
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• pp.1508-1513
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• 2006

Myostatin (MSTN) and insulin-like growth factors (IGFs) are a known inhibitor and stimulators of proliferation and differentiation of muscle cells, respectively. The present study was performed to investigate the relationship of MSTN-induced growth inhibition to expression of the IGF system components and myogenin, a muscle cell-specific transcription factor, in rat L6 myoblasts. The L6 cells transfected with a green fluorescent protein-MSTN plasmid expression construct had a 47% less cell number than mock-transfected cells after 3-d serum-free culture, accompanied by delayed differentiation which was suggested by inhibited aggregation of cells. Moreover, cells transfected with the expression construct had decreased expression of IGF-II and myogenin genes, but not IGF-I or its receptor genes, as examined by reverse transcription-polymerase chain reaction. The reduced mitosis of the L6 cells transfected with the MSTN-expression construct increased following an addition of either IGF-I or IGF-II to the culture medium, but not to the level of mock-transfected cells. By contrast, myogenin gene expression in these cells increased after the addition of either IGF to the level of mock-transfected cells. Collectively, these results suggest that the inhibitory effect of MSTN on L6 cell proliferation and differentiation is likely to be partly mediated by serially suppressed expression of IGF-II and myogenin genes, not IGF-I gene.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.sup3
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• pp.119-123
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• 2016

Thyroid cancer, the most common endocrine neoplasia, consists of four main types of carcinomas: papillary, follicular, and anaplastic, all with thyroid follicular origin, and medullary thyroid cancer (MTC) related to para-follicular cells. Cronic diseases such as diverse cancers may be associated with cachexia, especially at advanced stage. Cancer-induced cachexia is associated with diminished quality of life, functional performance, reduced response to antitumor therapy, and increased morbidity and mortality. Myostatin (Mst) is one of the outstanding molecules in the skeletal muscle loss process in cancer and it may be released by both skeletal muscle and cachexia-inducing tumors. Recently changes in serum levels of Mst have been identified as an important factor of cancer-induced cachexia. The goal of this study was to assessserum Mst levels in MTC patients. In this descriptive and case-control study, 90 participants were selected, comprising 45 MTC patients (20 males, $29{\pm}13.9years$, 25 females, $29{\pm}14.5years$) and 45 control individuals (25 males, $23.1{\pm}11.6years$, 20 females, $31.5{\pm}14.4years$). Serum Mst was determined using an ELISA kit and body mass index (BMI) was calculated by weight and height measurements. The Kolmogorov Simonov test showed a normal distribution for log transformed Mst serum levels in both case and control groups. Geometric means were 5.9 and 8.2 ng/ml respectively, and a significant difference was found according to the independent t-test results (P<0.01). There was also a significant difference mean of Mst between females in control and MTC groups, but not for the males. Pearson correlation test showed no correlation between age and BMI with Mst serum levels. The findings of this study support the hypothesis that Mst serum levels may have a potential ability for early diagnosis of cachexia in MTC patients, especially in females.

• Journal of Animal Science and Technology
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• v.47 no.2
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• pp.159-166
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• 2005

Porcine myostatin(MS1N) gene plays a key role in the differentiation of myoblast and muscle development. Genetic polymorphism was screened by single stranded conformation polymorphism(SSCP) analysis and subsequent DNA sequencing detected a nucleotide substitution(C2150T) in exon 3 of MSIN gene. Phenotypic association of the polymorphism was tested in a Landrace population and positive effects of the allele T for lean growth traits were found in the population. Even though it is not significant, the pigs have IT and TC genotypes were heavier for the body weight at birth and at twenty weeks of age than those containing genotype. Cc. However, the allele T was significantly associated with higher eye muscle area(P < 0.05). As a result of this study, we suggested that the allele T in exon 3 of MSTN gene comes a significant effect for increasing the eye muscle area without decreasing backfat thickness. This polymorphism did not change the amino acid but Taq I -RFLP matched to SSCP band patterns in exon 3 of MSTN gene, which will be an useful molecular marker for breeding of Landrace pigs.

• Journal of Acupuncture Research
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• v.31 no.1
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• pp.7-21
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• 2014

Objectives : To observe the regenerative effects of indirect moxibustion, a traditional Korean medical treatment on skeletal muscles using mouse model of skeletal muscle adiposity. Methods : Twenty seven ICR male mice were randomly assigned into Intact control(n=3), glycerol treatment together without moxibustion(n=12), and glycerol treatment together with moxibustion (n=12) groups. Mice of glycerol treatment groups were injected with 50 ${\mu}l$ DW(distilled water) containing 50 % of glycerol into the two tibialis anterior. After injection, moxibustion was applied at 'Shenshu'($BL_{23}$) and 'Zusanli'($ST_{36}$) acupoints three times per each session, every days for twelve days(total 12 treatments). Phospho-Erk1/2, Myostatin protein levels were analyzed by western blotting and immunofluo-rescence staining techniques for tissues of the tibialis anterior muscle. Smad, phospho-Smad were analyzed by immunofluorescence staining. Results : 1. Histological analysis of sections from injected TA muscles showed that glycerol induced rapidly muscle necrosis, with a maximum at day 3. 6 days and 9 days after injection, muscle was regenerating. 2. According to western blotting and immunofluorescence staining, phospho-Erk1/2 protein signals in glycerol treatment with moxibustion group were stronger compared to Intact and glycerol treatment without moxibustion group. 3. According to western blotting and immunofluorescence staining, myostatin protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. 4. According to immunofluorescence staining, Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. 5. According to immunofluorescence staining, phospho-Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. Conclusions : These results confirm that indirect moxibustion of 'Shenshu'($BL_{23}$) and 'Zusanli'($ST_{36}$) influences muscle regeneration in mouse models of skeletal muscle adiposity. Further discussion, and the establishment of moxibustion mechanism will prompt clinical application of moxibustion.

• Journal of Oral Medicine and Pain
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• v.45 no.4
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• pp.83-88
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• 2020

Purpose: Growth differentiation factor 11 (GDF11) and myostatin (MSTN) are closely-related transforming growth factor β family members reported to play crucial roles in bone formation. We previously reported that, in contrast to MSTN, GDF11 promotes osteogenesis of vertebrae and limbs. GDF11 has been also reported as an important regulator in tooth development by inducing differentiation of pulp stem cells into odontoblasts for reparative dentin formation. The goal of this study was to investigate the differential roles of GDF11 and MSTN in dental and cranial bone formation. Methods: Micro-computed tomography analysis was performed on cranial bones, including frontal, parietal, and interparietal bones, and lower incisors of wild-type, Gdf11 knockout (Gdf11-/-), and Mstn knockout (Mstn-/-) mice. Tissue volume, thickness, and mineral density were evaluated for both cranial bone and lower incisors. Lower incisor lengths were also measured. Because Gdf11-/- mice die shortly after birth, analysis was performed on newborn (P0) mice. Results: Compared to those of Mstn-/- mice, cranial bone volume, thickness, and mineral density levels were all significantly diminished in Gdf11-/- mice. Tissue mineral density of Gdf11-/- mice were also significantly decreased compared to wild-type mice. Likewise, lower incisor length, tissue volume, thickness, and mineral density levels were all significantly reduced in Gdf11-/- mice compared to Mstn-/- mice. Incisor length was also significantly decreased in Gdf11-/- mice compared to wild-type mice. Mstn-/- mice exhibited mildly increased levels of tissue volume, thickness, and density in cranial bone and lower incisor compared to wild-type mice although statistically not significant. Conclusions: Our findings suggest that GDF11, unlike MSTN, endogenously promotes cranial bone and tooth development.