华北农学报2017，Vol.32Issue(6)：25-30,6.DOI:10.7668/hbnxb.2017.06.004
简易型同源重组载体pAmpR-NeoR的构建和效果检测
Construction and Effect Test of Simple Homologous Recombination Vector pAmpR-NeoR
摘要
Abstract
In order to construct a donor plasmid that could be used for gene editing in CRISPR/Cas9 system, AmpR-ori fragment and NeoR fragment were amplified with pcDNA3. 1( +)-HA-His as template. And then these two fragments were ligated to construct a simple homologous recombination vector pAmpR-NeoR. As cloning sites, BamH Ⅰ and XhoⅠthat were introduced on both sides of the NeoR gene were used for the insrtion of the left and right homologous arms. Using this vector,we constructed a donor plasmid for targeting mouse FasL gene. The experi-mental results showed that the target sequences were genetically edited. Because both BamH Ⅰ and Xho Ⅰ had their own isocaudomers, pAmpR-NeoR had good scalability, flexibility and relative universality for sequence to be clonded,which was easy to further transform as needed to meet the different needs of specific experiments.关键词
基因编辑/同源重组载体/同尾酶Key words
Gene editing/Homologous recombination vector/Isocaudomers分类
生物科学引用本文复制引用
王英泽,王巧兰,曹晴晴,付育,刘畅,王晨晨,杜朝..简易型同源重组载体pAmpR-NeoR的构建和效果检测[J].华北农学报,2017,32(6):25-30,6.基金项目
国家自然科学基金面上项目(81171455) （81171455）
国家自然科学基金青年项目(31100715 （）
81402305) （）
河北省自然科学基金青年项目(C2013208005) （C2013208005）
河北省高等学校科学技术研究项目(QN2014043) （QN2014043）
河北科技大学五大平台开放基金课题 （）
中国科学院纳米生物效应与安全性重点实验室开放课题资助项目(NSKF201605) （NSKF201605）
