摘要
同源重组介导的基因打靶技术因其准确性高、引入的突变可以预测,已经成为功能基因组研究的重要工具。但是在真核生物体内,天然同源重组的概率极低,对后续的筛选和鉴别造成很大困扰。因此,建立一个广谱性的高效筛选策略极为重要。研究基于"双荧光"的可视化筛选,通过提高筛选效率,从而建立了一种显著提高同源重组打靶效率的新策略。该策略以绿色荧光蛋白基因和新霉素基因为正筛选标记,以红色荧光蛋白基因为负筛选标记。经过G418筛选后,挑取仅表达绿色荧光蛋白的抗性克隆,用于跨界PCR检测。研究尝试运用上述新策略,借助CRISPR/Cas9技术,对猪肌抑素基因进行同源重组介导的基因打靶。在选取的T1和T2两个靶位点处,"双荧光"策略筛选的细胞阳性率分别达到80.5%和86.7%,筛选效率得到显著提高。研究建立的"双荧光"可视化筛选策略具有高效性和广谱性,在动物基因编辑领域具有广阔的应用前景。
Gene targeting technology mediated by homologous recombination is a powerful tool for gene function analyze,because of its high accuracy and the mutation we introduced can be predicted. While in eukaryotes,the ratio of homologous recombination is too low to do further screening and identification. For this reason,establishing a high efficiency and widely used screening strategy becomes important. A new screening strategy which can dramatically improve the homologous recombination efficiency based on visual ＂double fluorescence＂screening was established. The green fluorescent protein and neomycin gene were used as positive selection markers,and red fluorescent protein gene for negative selection marker. After screening by G418,the cell clones which just expressing the green fluorescent protein gene were selected and detected by transboundary PCR. The new strategy was used to detecting the gene targeting of pig MSTN gene in pig cells combined with CRISPR / Cas9 technology. In the two target sites T1 and T2,the targeting efficiency screening by ＂double establishing＂reached up to 80. 5% and 86. 7%,which the was dramatically improved. This visual ＂double fluorescent＂screening strategy established will become a useful tool for animal gene editing in the future.
作者
陈建武
任红艳
华文君
刘西梅
綦世金
周黎
欧阳艳
毕延震
杨烨
郑新民
CHEN Jian-wu REN Hong-yan HUA Wen-jun LIU Xi-mei QI Shi-jin ZHOU Li OU Yang-yan BI Yan-zhen YANG Ye ZHENG Xin-min(College of Animal Sciences, Yangtze University, Jinzhou 434025, China Hubei Key Lab of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Science and Veterinary Hubei Academy of AgroSciences, Wuhan 430064, China College of Agriculture, Hubei Three Groges Vocational and Technical College, Yichang 443000, China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2017年第1期58-63,共6页
China Biotechnology
基金
转基因生物新品种培育科技重大专项(2016ZX08006001-005
2016ZX08006002-006
2016ZX08010003-006)
湖北省科技支撑计划(2014BBB010)
湖北省农业科技创新中心(2016-620-000-001-027)资助项目
