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.
出处
《华北农学报》
CSCD
北大核心
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)
关键词
基因编辑
同源重组载体
同尾酶
Gene editing
Homologous recombination vector
Isocaudomers
