利用CRISPR/Cas9系统构建Lrtm1基因敲除的C2C12细胞系

Construction of Lrtm1 gene knockout C2C12 cell line using the CRISPR/Cas9 system

摘要:

目的：利用CRISPR/Cas9技术构建稳定敲除Lrtm1（leucine-rich repeats and transmenbrane domains 1）基因的C2C12细胞系，为研究Lrtm1基因的作用提供实验基础。方法：设计3对针对Lrtm1基因的向导RNA（sgRNA），将sgRNA插入载体pCRISPR-LvSG06中；利用慢病毒包装系统包装含有sgRNA的重组质粒pCRISPR-LvSG06；将病毒感染C2C12细胞，并加入嘌呤霉素筛选，将筛选嘌呤霉素阳性的细胞提取RNA，逆转录成cDNA；设计Cas9引物，利用cDNA为模版，PCR验证C2C12细胞中Cas9的表达，确认慢病毒成功感染C2C12细胞；利用96孔板挑选单克隆细胞的方法筛选得到单克隆细胞；将扩增的单克隆细胞提取基因组DNA，测序Lrtm1基因相关序列并与野生型Lrtm1基因进行对比，确认敲除成功的克隆细胞株；诱导敲除Lrtm1稳定细胞株成肌分化，检测成肌分化标志因子Myosin的蛋白表达，RT-PCR检测转录因子PAX7的mRNA表达，Western blot检测H3K27me3蛋白水平。结果：测序结果显示向导RNA（sgRNA）成功插入载体质粒；将单克隆细胞DNA测序结果显示A和C克隆成功敲除Lrtm1基因；敲除Lrtm1基因后成肌分化标志因子Myosin蛋白表达降低，成肌转录因子PAX7 mRNA的表达降低，在分化72和96 h组，H3K27me3蛋白水平较野生型组增高。结论：利用CRISPR/Cas9技术成功敲除Lrtm1基因，稳定敲除Lrtm1基因的C2C12细胞系构建成功；敲除Lrtm1后能抑制C2C12细胞成肌分化，并且抑制成肌转录因子PAX7的mRNA表达，PAX7 mRNA表达降低的原因可能为H3K27me3水平增高。

Abstract:

Objective：To construct Leucine-rich repeats and transmembrane domains 1（Lrtm1） gene knockout C2C12 cell line using the CRISPR/Cas9 technique and to provide an experimental basis for studying the functions of the Lrtm1 gene. Methods：Three pairs of small guide RNA（sgRNA） targeting the Lrtm1 gene were designed and inserted into the vector pCRISPR-LvSG06，and the recom－binant plasmid pCRISPR-LvSG06 containing sgRNA was packaged by the lentiviral packaging system. Then the C2C12 cells were infected with the virus and screened by puromycin，and RNA was extracted from the screen-positive cells and reversely transcribed into cDNA. The Cas9 primer was designed using the cDNA as the template to verify the expression of Cas9 in C2C12 cells by PCR and to confirm that the C2C12 cells were successfully infected by the virus. Monoclonal cells were collected by the 96-well plate method，and the amplified monoclonal cells were used to extract genomic DNA and investigate the Lrtm1 gene-related sequences for comparison with the wild-type Lrtm1 gene，so as to identify the monoclonal cell line with successful gene knockout. After that，the stable Ltm1 gene knockout cell line was induced to differentiate into myoblasts and its expression of myosin，which was the marker of successful myogenic differentiation，was determined. Besides，the expression of transcription factor PAX7 mRNA and H3K27me3 protein was determined by RT-PCR and Western blot，respectively. Results：The sequencing results showed the successful insertion of the sgRNA into the plasmid vector and Lrtm1 gene knockout in clones A and C. The expression of both myosin protein as the myogenic differentiation marker and PAX7 mRNA as the myo－genic transcription factor decreased，while the expression levels of H3K27me3 protein in both the 72-hour and 96-hour differ－entiation groups were higher than those in the wild-type group. Conclusion：The CRISPR/Cas9 technique can be used for Lrtm1 gene knockout and successfully constructing the Lrtm1 gene knockout C2C12 cell line. Lrtm1 gene knockout can inhibit the myogenic dif－ferentiation of C2C12 cells and the expression of PAX7 mRNA，and the reason for the reduced expression of PAX7 mRNA may be at－tributed to the increased level of H3K27me3.