摘要
目的建立敲除基因组中PDE10A基因的CRISPR/Cas9系统。方法设计3个长20bp的sgRNA,分别靶向PDE10A的exon 6、exon 7和exon 11。化学合成sgRNA寡核苷酸序列,并克隆进PX330质粒中。将克隆正确的质粒PX330-sgRNA转染至HEK293T细胞中,提取基因组DNA并对敲除位点附近的DNA片段进行PCR扩增,再通过SURVEYOR分析和一代测序对敲除效率进行检测。最后采用有限稀释法挑选稳定敲除PDE10A的HEK 293T细胞株。结果目的sgRNA寡核苷酸双链成功插入PX330质粒中且序列正确;靶向Exon 7的sgRNA可成功敲除PDE10A,且敲除效率高达31.4%;稳定敲除PDE10A的细胞株筛选成功,可导致2 bp缺失。结论PDE10A基因CRISPR/Cas9敲除系统构建成功。
Objective To construct the CRISPR/Cas9 system constructed by knocking out the PDEIOA gene. Methods Three 20 bp sgRNAs targeting exon 6, exon 7 and exon 11 of PDEIOA were designed, chemically synthesized, and then inserted into linearized plasmid, PX330. Next, the correct PX330-sgRNA plasmids were transfected into HEK293T cells after verification by Sanger sequencing. The targeting efficiency was detected by SURVEYOR assay and the nicked site was further detected by Sanger sequencing. Finally, the stable PDEIOA knockout 293T cell line was selected by the lim- iting dilution method. Results The target nucleotide sequences were successfully inserted into the expected sites of vec- tor and sequences were correct. The targeted exon 7 sgRNA can successfully knock PDEIOA, and the targeting efficiency was up to 31.4 %. Stable knockout PDEIOA cell line was selected successfully which harbored 2 bp deletion. Conclu- sions The CRISPR/Cas9 system constructed by knocking out the PDEIOA gene is successfully constructed.
出处
《基础医学与临床》
CSCD
北大核心
2014年第4期439-443,共5页
Basic and Clinical Medicine
基金
国家自然科学基金(31222031)
中央高校基本科研业务费专项资金(2012S05)
协和青年科研基会(2012J09)
新世纪优秀人才支持计划(NCET-12-0071)
