敲除L-periaxin基因的大鼠RSC96细胞系的建立

Establishment of L-periaxin gene knock-out RSC96 cell line

摘要:

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。

Abstract:

Periaxin, a protein of noncompact myelin, is specifically expressed in the peripheral nervous system (PNS). There are two protein isoform L-periaxin and S-Periaxin by alternative splicing of periaxin gene, playing an important role in the initiation of myelin formation. So far, 18 different mutation sites in L-periaxin gene have been found to induce the peripheral demyelinating neurological charcot-marie-tooth diseases subtype 4F (CMT4F). The technique of activation of transcription activator-like effector nucleases (TALENS) was used to knock out the L-periaxin gene in RSC 96 cell line of Rattus. According to the design principle, the knock-out site of L-periaxin was assured to NLS domain of L-periaxin, which is target sequence of left and right arms of TALEN. The knock-out vectors of TALEN-L and TALEN-R were established and transfected into RSC96 cell. After puromycin screening, L-periaxin was knocked out successfully in RSC96 cell, which is confirmed by DNA sequence. The mutation efficiency is 21.6%. S-periaxin, not L-periaxin can be detected by Western blotting in L-periaxin gene knock-out RSC96 cell. The cell growth rate was decreased and the number of cells in G1 increased and decreased in S phase in L-periaxin gene knock-out RSC96 cell by flow cytometry and MTT assay.