生命科学研究2018，Vol.22Issue(4)：277-282,6.DOI:10.16605/j.cnki.1007-7847.2018.04.003
基于CRISPR/Cas9系统的人成神经瘤SK-N-SH细胞CAPNS1基因的靶向敲除
Targeting Knockout of CAPNS1 Gene in SK-N-SH Human Neuroblastoma Cells Based on CRISPR/Cas9 System
摘要
Abstract
SK-N-SH human neuroblastoma cell line targeting CAPNS1 gene was constructed by using CRISPR/Cas9 technology. The CDS region of the gene was obtained by using the NCBI database, and according to the principles for designing the CRISPR/Cas9 knockout target site (g RNA), three sg RNAs were designed, and the knockout vector was constructed by using p GK1.1 as a vector. The recombinant colonies were confirmed positive as the 100 bp fragment could be amplified from them by PCR. DNA sequencing and sequence alignment showed that sg RNA was correctly inserted into the plasmid. The plasmid was then transfected into SK-N-SH cells and single cell colonies were prepared. The suspected positive clones were screened by CruiserTMEnzyme. Clone sequencing further confirmed that the CAPNS1 gene was deleted from the SK-N-SH cells. In addition, CAPNS1 protein, calpain1 and calpain2 protein levels were significantly reduced in CAPNS1-/-group. These results demonstrated the CAPNS1 knockout cell line of SK-N-SH was successfully constructed.关键词
CRISPR/Cas9系统/CAPNS1/基因敲除/SK-N-SH细胞Key words
CRISPR/Cas9/CAPNS1/gene knockout/SK-N-SH cells分类
生物科学引用本文复制引用
朱家佳,龙鼎新..基于CRISPR/Cas9系统的人成神经瘤SK-N-SH细胞CAPNS1基因的靶向敲除[J].生命科学研究,2018,22(4):277-282,6.基金项目
国家自然科学基金资助项目(81673227, 81172712) （81673227, 81172712）
