期刊论文

Targeting Knockout of CAPNS1 Gene in SK-N-SH Human Neuroblastoma Cells Based on CRISPR/Cas9 System

中文

生命科学研究

1007-7847

2018

22

4

277-282

10.16605/j.cnki.1007-7847.2018.04.003

81673227:国家自然科学基金 81172712:国家自然科学基金

本校为第一机构

利用CRISPR/Cas9技术靶向构建CAPNS1基因敲除人成神经瘤细胞SK-N-SH细胞系。在NCBI数据库查找人CAPNS1基因,获得该基因CDS区,根据CRISPR/Cas9敲除靶位点设计原则即g RNA设计原则,设计3条sg RNAs,以p GK1.1为载体,构建人CAPNS1基因敲除载体,并运用共转染的方法转染到SK-N-SH细胞,构建CAPNS1基因敲除SK-N-SH细胞系。结果发现,菌落PCR筛选均能扩增出100 bp大小的片段,单克隆为阳性,DNA测序显示sg RNA序列正确插入质粒,序列比对结果正确;质粒转染SK-N-SH细胞后,制备单克隆,CruiserTMEnzyme酶切后疑似为阳性克隆,进一步的单克隆测序结果显示CAPNS1基因敲除SK-N-SH细胞系构建成功;此外,在CAPNS1-/-组,...

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 (gRNA), three sgRNAs were designed, and the knockout vector was constructed by using pGK1.1 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 sgRNA was correctly inserted into the plasmid. The plasmid was then transf...