• Asian-Australasian Journal of Animal Sciences
• /
• v.30 no.7
• /
• pp.1029-1036
• /
• 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.

• Journal of Animal Reproduction and Biotechnology
• /
• v.37 no.4
• /
• pp.292-297
• /
• 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.

• The Journal of Korean Academy of Prosthodontics
• /
• v.18 no.1
• /
• pp.73-86
• /
• 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
• /
• v.7 no.1
• /
• pp.12-15
• /
• 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
• /
• v.21 no.2
• /
• pp.206-212
• /
• 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
• /
• v.29 no.7
• /
• pp.1037-1043
• /
• 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.

• The Korean Journal of Vision Science
• /
• v.20 no.4
• /
• pp.569-577
• /
• 2018

Purpose : The study looked at how effective each group wearing MyoVison lens, MC lens, and Single Vision lensdepending on their parents' myopia condition. Methods : The study observed the changeof spherical equivalent among customers, who visited between January 2010 and December 2016,of an optical shop in Incheon Metropolitan City. And we observed MyoVision 152 eyes, MC Lens 86 eyes and Single Vision lens 270 eyes. This study was conducted using SPSS ver18, which analyzes the changes in average values of MyoVision, MC Lens, and Single Vision for a year.In each group, the differences in the group were compared using the Paired T-test and then one-way ANOVA (post-hoc; Bonferroni) Results : Group-to-group comparisons showed that MyoVision and MC Lens have a shorterinhibition than Single Vision. In particular, MyoVisionand MC Lens showed different relief effects depending on the degree of refraction of parents.When both parents had normal refractive, the change between MyoVision and Single Vision lens was $-0.35{\pm}0.05D$. When the father had a refraction MC lens were $-0.36{\pm}0.14D$ more effective than Single Vision. When only the mother had refraction, the mean value between MyoVision and Single Vision lens was $-0.37{\pm}0.06D$, and the mean between MC lens and Single Vision lens was $-0.38{\pm}0.08D$. And when both parents had refraction problems, the mean value change between MyoVision and Single Vision lens was $-0.28{\pm}0.07D$, and $-0.31{\pm}0.07D$, respectively. Conclusion : MyoVision and MC Lens appeared to have no effect on the functions of mitigating myopia in within group comparisons, but MyoVision and MC Lens showed reducing myopia than Single Vision in between group.

• Journal of Microbiology
• /
• v.42 no.1
• /
• pp.20-24
• /
• 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.

• Journal of Embryo Transfer
• /
• v.25 no.1
• /
• pp.1-7
• /
• 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.

• Microbiology and Biotechnology Letters
• /
• v.46 no.2
• /
• pp.102-110
• /
• 2018

A bacterial strain capable of metabolizing myo-inositol (MI) and converting to other substances was isolated from soil of orchard. The isolate, named YB-46, was grown on minimal medium supplemented with MI as the sole carbon source and was presumed to belonging to genus Enterobacter according to the 16S rDNA sequence. Escherichia coli transformant converting MI into unknown metabolites was selected from a metagenomic library prepared with fosmid pCC1FOS vector. Plasmid was isolated from the transformant, and the inserted gene was partially sequenced. From the nucleotide sequence, an iolG gene was identified to encode myo-inositol dehydrogenase (IolG) consisting of 336 amino residues. The IolG showed amino acid sequence similarity of about 50% with IolG of Enterobacter aerogenes and Bacillus subtilis. The His-tagged IolG (HtIolG) fused with hexahistidine at C-terminus was produced and purified from cell extract of recombinant E. coli. The purified HtIolG showed maximal activity at $45^{\circ}C$ and pH 10.5 with the highest activity for MI and D-glucose, and more than 90% of maximal activity for D-chiro-inositol, D-mannitol and D-xylose. $K_m$ and $V_{max}$ values of the HtIolG for MI were 1.83 mM and $0.724{\mu}mol/min/mg$ under the optimal reaction condition, respectively. The activity of HtIolG was increased 1.7 folds by $Zn^{2+}$, but was significantly inhibited by $Co^{2+}$ and SDS.