| Abstrakt: |
To study the role of chromosomeseegregation 1-like (CSE1L) protein in skeletal muscle development in mice, CSE1L gene knockout C2C12 cell line was constructed by CRISPR-Cas9 gene editing technology. 3 pairs of single RNAs (sgRNAs) targeting the third exon of mouse CSE1L CDS sequence were designed, then sgRNA plasmid (linked to green fluorescent protein, GFP) and Cas9 plasmid (linked to red fluorescent protein, RFP) were constructed. The sgRNA and Cas9 were co-transfected into C2C12 cell line, then the cleavage efficiency of different sgRNA was screened. After co-transfection, the C2C12 cell lines were sorted by flow cytometry to obtain the cells coexpressing GFP and RFP, and then identified by DNA sequencing. The molecular characteristics of CSE1L were identified by real-time quantitative PCR (RT-qPCR) and Western blot. Finally, CCK-8, cell cycle and apoptosis test were used to examine the activity and apoptosis of CSE1L knock out cell line. The results showed that CSE1L gene mRNA and protein expression level were significantly decreased (P<0.01), which indicated that CSE1L gene was successfully knocked out. CSE1L gene deletion resulted in C2C12 cell line elongated, cell viability was significantly down-regulated at 72 h, cell cycle arrest and apoptosis increased. This study demonstrated the CSE1L gene knockout cell line was successfully constructed, and the function related to growth and development of mouse skeletal muscle cells was studied, which provided a cell line model for exploring the function of CSE1L gene study and laid a foundation for generating subsequent gene editing knockout mice. [ABSTRACT FROM AUTHOR] |
