• Asian-Australasian Journal of Animal Sciences
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• 제30권7호
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• pp.1029-1036
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• 2017

Objective: In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation can be applied to improve traits such as growth and meat production. We investigated the regulatory pathway of MyoD and its role in muscle differentiation in quail myoblast cells. Methods: The MyoD gene was mutated by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology and single cell-derived MyoD mutant sublines were identified to investigate the global regulatory mechanism responsible for muscle differentiation. Results: The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (MyoD KO QM7#4) through single cell pick-up and expansion. In the undifferentiated condition, paired box 7 expression in MyoD KO QM7#4 cells was not significantly different from regular QM7 (rQM7) cells. During differentiation, however, myotube formation was dramatically repressed in MyoD KO QM7#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD KO QM7#4 cells even after an extended differentiation period. These results indicate that MyoD is critical for muscle differentiation. Furthermore, we analyzed the global regulatory interactions by RNA sequencing during muscle differentiation. Conclusion: With CRISPR/Cas9-mediated genomic editing, single cell-derived sublines with a specific knockout gene can be adapted to various aspects of basic research as well as in functional genomics studies.

• 한국동물생명공학회지
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• 제37권4호
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• pp.292-297
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• 2022

Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach.

• 대한치과보철학회지
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• 제18권1호
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• pp.73-86
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• 1980

Recently, the controversy continues as to whether maximum intercuspation of teeth should occur at the terminal hinge position(the condylar theory) or at the myo-co(the neuromuscular theory). There is also much controversy regarding the antero-posterior position of myo-co. The object of this study was to measure and compare with the positional relations of centric relation, centric occlusion and myo-co, and free-way space using Mandibular Kinesiograph and Myo-monitor in the 40 subjects without stomatognathic problems. Mandibular Kinesiograph(M.K.G.) was originally conceived as a research instrument to track mandibular movement and position. As its use in research progressed, its great diagnostic value became apparent in case by case. And Myo-monitor was developed as a means of applying the neuromuscular approach to occlusion. Thus the Myo-monitor technique is an intra-systemic approach to occlusal positioning using patient's own musculature, and Myo-monitor is used to relax the musculature by a light myopulse induced electronically. From this experiment, the following results were obtained. 1. The adaptive free-way space before muscle relaxation was an average of $1.6{\pm}60mm$, and the true free-way space after muscle relaxation using Myo-monitor was an average of $2.4{\pm}0.74mm$. 2. It took an average of $25{\pm}3.11$ minutes to relax the mandibular musculature by Myo-monitor and administration of 5mg. Diazepam and an average of $38{\pm}4.73$ minutes by Myo-monitor without administration of Diazepam. 3. Myo-co existed anterior to centric occlusion, with an average of $0.53{\pm}0.31$ mm, and centric relation existed posterior to centric occlusion, with an average of $0.57{\pm}0.58mm$ before muscle relaxation and with an average of $0.57{\pm}0.43mm$ after muscle relaxation. 4. Centric relation coincided with centric occlusion in 5 of 40 subjects(12.5%), and posterior to centric occlusion in the rest of cases (87.5%). 5. Myo-co existed anterior to centric occlusion in 38 of 40 subjects(95%), except 1 subject that coincided with centric occlusion and 1 subject that existed posterior to centric occlusion. 6. Myo-co and centric relation existed inferior to centric occlusion and the lateral displacement was various with individual difference. 7. The total displacement from centric occlusion to centric relation was an average of $0.74{\pm}0.64mm$ before muscle relaxation, and an average of $0.68{\pm}0.53mm$ after muscle relaxation, and the total displacement from centric occlusion to myo-co was an average of $1.07{\pm}0.58mm$.

• Mass Spectrometry Letters
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• 제7권1호
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• pp.12-15
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• 2016

Results of free and bound myo-inositol in infant formula (IF) are presented. Inositol was analyzed by HILIC ultra-performance liquid chromatography coupled with mass spectrometer. The levels of free myo-inositol in 27 Australian and 4 EU originated IF samples were 300-600 mg/kg of powder or 1.6-3.1 mg/100 kJ. The amount of bound inositol in lipid fraction of IF was, on average, 10% of free myo-inositol.

• Molecules and Cells
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• 제21권2호
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• pp.206-212
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• 2006

We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and $p16^{INK4a}$ functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and $p16^{INK4a}$ pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.

• Asian-Australasian Journal of Animal Sciences
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• 제29권7호
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• pp.1037-1043
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• 2016

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

• 대한시과학회지
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• 제20권4호
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• pp.569-577
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• 2018

목적 : 본 연구는 부모의 근시정도에 따라 MC lens, MyoVisonlens 그리고 Single Vision lens를 착용한 그룹들이 각각 어느 정도 효과를 보이는지를 알아보고자 하였다. 방법 : 연구대상은 인천광역시 소재의 한 안경원의 고객 중 2010년 1월에서 2016년 12월 사이에 방문한 7세에서 20세 사이 안질환이 없는 근시안 MyoVision 152안, MC lens 86안, Single Vision lens 270안을 대상으로 등가 구면도수의 변화를 관찰하였다. 본 연구 자료는 MyoVision, MC lens 그리고 Single Vision의 1년간 평균값 변화를 SPSS ver18을 이용하여 분석하였다.각 그룹별로 처음 방문했을 때와 1년 후 재방문 했을 때의 그룹 내 차이를 Paired T-test 이용하여 비교하였고, 이후 One-Way ANOVA(Post Hoc; Bonferroni)분석을 통해 사후분석을 진행하였다. 결과 : 그룹간 비교에서는 MyoVision과 MC lens가 Single Vision 보다 근시 완화효과가 있는 것으로 나타났다. 특히 부모의 굴절이상도에 따라 MyoVision과 MC lens의 근시 완화효과가 다르게 나타났다. 부모 모두 굴절 이상도가 정상일 때는 MyoVision 과 Single Vision lens 간의 변화는 $-0.35{\pm}0.05D$로 통계적으로 유의한 변화값이 나타났다. 아버지만 굴절이상이 있을 때는 MC lens는 Single Vision 보다 $-0.36{\pm}0.14D$만큼 더 효과적인 것으로 나타났다. 그리고 어머니만 굴절이상이 있을 때는 MyoVision 과 Single Vision lens 간의 평균값은 $-0.37{\pm}0.06D$의 변화를 보였고 MC lens 와 Single Vision lens 간의 평균값은 $-0.38{\pm}0.08D$가 변화하여 각각 Single Vision lens에 비해서 근시를 완화시켜주는 효과가 있는 것으로 나타났다.부모모두 굴절이상이 있을 때는 MyoVision 과 Single Vision lens 간의 평균값 변화와 MC lens 와 Single Vision lens 간의 평균값 변화는 각각 $-0.28{\pm}0.07D$, $-0.31{\pm}0.07D$로 MyoVision 과 MC렌즈 모두 통계적으로 유희한 변화값을 보여 근시진행에 억제 효과가 있는 것으로 나타났다. 결론 : 본 연구결과 각 그룹 내에서 MyoVision, MC lens 그리고 Single Vision모두 근시를 완화시켜주는 기능에 있어서 효과가 없는 것으로 보였으나 그룹 간 비교에서는 MyoVision과 MC lens가 Single Vision 보다 근시 완화효과가 있는 것으로 나타났다.

• Journal of Microbiology
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• 제42권1호
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• pp.20-24
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• 2004

Cloned myo-inositol-1-phosphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His-affinity column. The purified INOS required NAD$\^$+/ for the conversion of glucose-6-phosphate to inositol-1-phosphate. The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40$^{\circ}C$. The molecular weight of the native enzyme, as determined by gel filtration, was approximately M$\_$r/ 271,000${\pm}$15,000. A single subunit of approximately M$\_$r/ 62,000${\pm}$5,000 was detected upon SDS-polyacrylamide gel electrophoresis. The Michaelis ($K_{m}$) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD$\^$+/ these were 0.42 and 0.4 mM, respectively.

• 한국수정란이식학회지
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• 제25권1호
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• pp.1-7
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• 2010

The objective of this study was to examine the effect of vitamin B (pantothenic acid, folic acid, and myo-inositol) that was supplemented to embryo culture medium on in vitro development of parthenogenetically activated (PA) pig embryos. Cumulus-oocyte complexes derived from slaughtered ovaries were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (hCG and eCG) for the first 22 h and then further cultured in hormone-free medium for an additional 22 h. After maturation culture, metaphase II oocytes that extruded 1st polar body were electrically activated and treated with $5.0\;{\mu}g/ml$ cytochalasin B for 4 h. Then, PA embryos were cultured for 7 days in a modified NCSU-23 that was supplemented with pantothenic acid, myo-inositol, or folic acid at different concentrations ($3{\sim}300\;{\mu}M$) according to the experimental design. Myo-inositol added to culture medium did not show any beneficial or inhibitory effects on embryo cleavage and blastocyst formation. However, $300\;{\mu}M$ pantothenic acid significantly inhibited blastocyst formation compared to control (no addition) (24% vs. 36%, p<0.05). Folic acid ($300\;{\mu}M$) significantly (p<0.05) increased blastocyst formation (56%) compared to control (41%). Our results demonstrated that in vitro development of PA embryos was significantly influenced by vitamin B and addition of $300\;{\mu}M$ folic acid to culture medium improved in vitro development of pig PA embryos.

• 한국미생물·생명공학회지
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• 제46권2호
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• pp.102-110
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• 2018

myo-Inositol (MI)을 대사하여 다른 물질로 전환하는 미생물을 과수원 토양으로부터 분리하였다. 분리균 YB-46은 유일한 탄소원으로 MI이 첨가된 배지에서 성장하였고 16S rDNA 염기서열에 따라 Enterobacter 속의 균주로 추정되었다. Fosmid pCC1FOS 벡터를 사용하여 제조된 거대 유전체 은행으로부터 MI을 미지의 대사 물질로 전환하는 Escherichia coli 형질전환주를 선발하였다. 이로부터 플라스미드를 분리하고 삽입된 유전자의 일부 염기서열을 결정한 결과 336 아미노 잔기로 구성된 myo-inositol dehytrogenase (IolG)를 암호화하는 iolG 유전자가 발견되었다. 분리균 YB-46의 IolG는 E. aerogenes와 Bacillus subtilis의 IolG와 약 50% 수준의 상동성을 보였다. 카르복실 말단에 hexahistidine이 연결되도록 제조한 His-tagged IoG (HtIolG)의 유전자를 재조합 대장균에서 발현하여 균체 파쇄액으로부터 HtIolG를 정제하였다. 정제된 HtIolG는 $45^{\circ}C$와 pH 10.5에서 최대 활성을 보였고 MI과 D-glucose에 대한 활성이 가장 높았으며 D-chiro-inositol, D-mannitol 및 D-xylose에도 90% 이상의 활성을 보였다. 최적 반응조건에서 MI을 기질로 하여 반응 동력학적 계수를 측정한 결과 $K_m$과 $V_{max}$가 1.83 mM과 $0.724{\mu}mol/min/mg$로 확인되었다. HtIolG의 활성은 $Zn^{2+}$에 의해 1.7배 증가하였으며, $Co^{2+}$와 SDS에 의해서는 크게 감소하였다.