Study on Muscle Differentiation in Quail Myoblast Cells Genome-Edited by CRISPR-Cas9 System

Abstract

In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation are very important to understand skeletal muscle growth. This study is about to investigate the regulatory pathway of MyoD and its role in muscle differentiation in QM7 (quail muscle clone 7) myoblast cells. 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 regulatory mechanism responsible for muscle differentiation. We induced the differentiation by changing medium without serum during cell culture. The mutation efficiency in the mixed population was 73.3%. We selected a single cell (MyoD KO QM7#4) from the mixed population and cultured to expansion. We confirmed the difference between on the 3, 6 day of differentiation. The expression of paired box 7 (Pax7), which is the undifferentiated marker, was not significantly different from regular QM7 (rQM7) cells to MyoD KO QM7#4 cells. However, there was a significant difference in morphology between the rQM7 cells and MyoD KO QM7#4 cells during differentiation. The differentiation to myotube formation and the nuclear fusion rate was glaringly suppressed in MyoD KO QM7#4 cells comparing with rQM7 cells. And the myogenic differentiation-related genes also were not detected in MyoD KO QM7#4 cells during differentiation. In conclusion, we can assume that MyoD is a critical factor for muscle differentiation in the quail myoblast and the CRISPR/Cas9-mediated genomic editing can be adapted to various aspects, not only for this study, but also for functional genomic study in the poultry science.

Keywords : myoblast, CRISPR-Cas9, knockout, muscle differentiation, MyoD Student Number: 2015-22418