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轴周蛋白(Periaxin)是外周神经系统非致密性髓鞘中特异表达的蛋白,编码Periaxin的基因经由选择性剪接产生两种蛋白异构体L-periaxin和S-periaxin,对髓鞘形成的初始化有重要作用。至今在Periaxin基因上已发现有18种不同的位点突变导致外周脱髓鞘神经疾病腓骨肌萎缩症4F亚型(CMT4F)的发生。利用转录激活因子样效应物核酸酶(TALENs)靶向基因敲除技术对大鼠RSC96细胞的periaxin基因进行敲除,根据TALENs设计原则,确定L-periaxin基因的敲除靶点在其编码NLS结构域部分,设计相应的TALEN左臂与右臂的识别序列,构建含上述识别序列的L-periaxin基因敲除载体TALEN-L和TALEN-R,并将其转入RSC96细胞,经嘌呤霉素药物筛选,获得L-periaxin基因敲除细胞株,经测序确认大鼠RSC96细胞中的基因组中L-periaxin基因区段已被敲除,成功构建了L-periaxin基因敲除细胞模型。计算L-periaxin基因敲除载体的突变率为21.6%。Western blotting实验证明,在RSC96细胞中只能检测到S-periaxin蛋白的表达。通过流式细胞术及MTT实验检测基因敲除细胞的细胞周期和生长速度,发现敲除L-periaxin基因的细胞生长速度缓慢,G1期细胞增多,S期细胞减少。
Periaxin is a protein specifically expressed in the non-myelin sheath of the peripheral nervous system. The genes encoding Periaxin produce two protein isoforms, L-periaxin and S-periaxin, via alternative splicing, initiating myelination Have an important role. To date, 18 different site mutations have been found in the Periaxin gene, leading to the occurrence of Charcot-Marie-Tooth Disease 4F subtype (CMT4F) in peripheral demyelinating neuropathy. The periaxin gene of rat RSC96 cells was knocked out by using TALENs targeting gene knockout technology. According to the design principles of TALENs, the knockout target of L-periaxin gene was identified in its encoding NLS structure Domain, designed corresponding TALEN left arm and right arm recognition sequence, construct L-periaxin gene knockout carrier TALEN-L and TALEN-R containing the above recognition sequence, and transferred into RSC96 cells, puromycin drug The L-periaxin gene knockout cell line was screened and the L-periaxin gene knockout cell model was successfully constructed by sequencing the L-periaxin gene segment of the rat RSC96 cell line was knocked out. The mutation rate of L-periaxin gene knockout vector was calculated to be 21.6%. Western blotting experiments showed that only the expression of S-periaxin protein was detected in RSC96 cells. The cell cycle and growth rate of knockout cells were detected by flow cytometry and MTT assay. It was found that the cell growth rate of L-periaxin gene knockdown was slow, the number of cells in G1 phase increased, and the number of S phase cells decreased.