基于CRISPR-Cas9技术建立CSE1L基因敲除C2C12细胞

Establishment of CSE1L Knockout C2C12 Cells by CRISPR-Cas9 System

为研究染色体分离样蛋白1(chromosomesegregation 1-like,CSE1L)在小鼠骨骼肌发育中的作用,利用CRISPR-Cas9基因编辑技术构建CSE1L基因敲除的C2C12细胞系。针对小鼠CSE1L的第3外显子设计3组sgRNA,构建sgRNA表达质粒(连接绿色荧光蛋白,green fluorescent protein,GFP)和Cas9质粒(连接红色荧光蛋白,red fluorescent protein,RFP)共转染C2C12细胞,筛选高切割效率的sgRNA;将转染该sgRNA和Cas9的C2C12细胞进行流式细胞分选,获取共表达红、绿双色荧光蛋白的细胞,并用DNA测序进行鉴定。通过实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹(Western-blot)鉴定其分子特征。最后分别利用CCK-8、细胞周期以及凋亡实验对CSE1L敲除细胞系活力和凋亡进行检测。结果显示,小鼠成肌细胞中CSE1L基因mRNA和蛋白水平表达量都显著降低(P<0.01),提示CSE1L基因敲除成功。该基因缺失导致C2C12细胞变细长、细胞活力在72 h显著下调,细胞周期停滞、凋亡增加。成功构建了CSE1L基因敲除的细胞系,并研究了小鼠骨骼肌细胞生长发育相关功能,为探索CSE1L基因功能提供细胞模型,并为后续基因编辑敲除鼠制备奠定基础。

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 co�expressing 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.