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Asian-Australas J Anim Sci > Accepted
doi: https://doi.org/10.5713/ajas.16.0749    [Accepted]
Myotube differentiation in CRISPR/Cas9-mediated MyoD knockout quail myoblast cells
Si Won Kim, Jeong Hyo Lee, Byung-Chul Park*, Tae Sub Park*
Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do 25354, Korea
* Corresponding Author: Byung-Chul Park ,Tel: 033-339-5721, Fax: 033-339-5763, Email: taesubpark@snu.ac.kr / Tae Sub Park ,Tel: 033-339-5721, Fax: 033-339-5763, Email: taesubpark@snu.ac.kr
Received: September 29, 2016;  Revised: October 8, 2016.  Accepted: September 29, 2016.  Published online October 28, 2016.

ABSTRACT
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.
Method:
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. The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (QM7 MyoD KO #4) through single cell pick-up and expansion. In the undifferentiated condition, Pax7 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 QM7 KO#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD QM7 KO#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.
Keywords: myoblast; CRISPR-Cas9; knockout; muscle differentiation; MyoD
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