Kim Si Won, Lee Jeong Hyo, Park Byung-Chul, Park Tae Sub
Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea.
Asian-Australas J Anim Sci. 2017 Jul;30(7):1029-1036. doi: 10.5713/ajas.16.0749. Epub 2016 Oct 28.
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.
The 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.
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.
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.
在畜牧业中，肌肉增殖和分化的调控机制可用于改善生长和产肉等性状。我们研究了MyoD的调控途径及其在鹌鹑成肌细胞肌肉分化中的作用。
利用成簇规律间隔短回文重复序列（CRISPR）/Cas9技术对该基因进行突变，并鉴定单细胞来源的MyoD突变亚系，以研究负责肌肉分化的整体调控机制。
混合群体中的突变效率为73.3%，通过单细胞挑选和扩增，我们从该群体中建立了两个QM7 MyoD基因敲除亚系（MyoD KO QM7#4）。在未分化状态下，MyoD KO QM7#4细胞中配对盒7的表达与正常QM7（rQM7）细胞无显著差异。然而，在分化过程中，MyoD KO QM7#4细胞中的肌管形成受到显著抑制。此外，即使在延长分化期后，MyoD KO QM7#4细胞中也未表达成肌分化特异性转录本和蛋白质。这些结果表明MyoD对肌肉分化至关重要。此外，我们在肌肉分化过程中通过RNA测序分析了整体调控相互作用。
通过CRISPR/Cas9介导的基因组编辑，具有特定敲除基因的单细胞来源亚系可应用于基础研究的各个方面以及功能基因组学研究。
