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BamH Ⅰ和Xho Ⅰ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。

关键词: 基因编辑, 同源重组载体, 同尾酶

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 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.

Key words: Gene editing, Homologous recombination vector, Isocaudomers

中图分类号: 

• Q78