Establishment of L-periaxin gene knock-out RSC96 cell line.
- Author:
Min LIANG
1
;
Tingting PENG
1
;
Yawei SHI
1
Author Information
- Publication Type:Journal Article
- Keywords: RSC96 cell; TALENs; gene knock-out; periaxin
- MeSH: Animals; Cell Line; Charcot-Marie-Tooth Disease; genetics; Gene Knockout Techniques; Membrane Proteins; genetics; Mutation; Protein Isoforms; Rats
- From: Chinese Journal of Biotechnology 2016;32(12):1735-1744
- CountryChina
- Language:Chinese
- 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.
