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

作　　者：王英泽[1] 王巧兰 曹晴晴 付育[1] 刘畅[1] 王晨晨 杜朝[1] 

机构地区：[1]河北科技大学生物科学与工程学院,河北石家庄050018

出　　处：《华北农学报》2017年第6期25-30,共6页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金面上项目(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);河北省高等学校科学技术研究项目(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)

摘　　要：为了构建能够灵活用于CRISPR/Cas9系统进行基因编辑的供体质粒,利用pcDNA3.1(+)-HA-His为模板,分别扩增了其AmpR-ori片段和NeoR片段,然后连接,构建了一个简易型同源重组载体pAmpR-NeoR。载体的正选择标记NeoR基因两侧分别引入了BamHⅠ和XhoⅠ作为克隆位点,用于左、右同源臂的克隆。利用这一载体,构建了针对小鼠FasL基因的供体质粒。结果显示靶序列发生基因编辑。pAmpR-NeoR以同尾酶作为多克隆位点,这使其具有良好的扩展性、灵活性和序列通用性,便于根据需要进一步改造,以满足具体试验的不同需求。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 Xho Ⅰ that 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 experimental 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 

分 类 号：Q78[生物学—分子生物学]