一种用于提高基因打靶效率的双荧光筛选策略

发布时间：2018-04-06 20:17

本文选题：基因打靶　切入点：CRISPR/Cas　出处：《中国生物工程杂志》2017年01期

【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。
[Abstract]:Homologous recombination mediated gene targeting has become an important tool for functional genome research because of its high accuracy and predictive mutation.However, in eukaryotes, the probability of natural homologous recombination is very low, which is a major problem for subsequent screening and identification.Therefore, it is very important to establish a broad spectrum efficient screening strategy.The visual screening based on "double fluorescence" was studied. By improving the screening efficiency, a new strategy to improve the efficiency of homologous recombination shooting was established.In this strategy, green fluorescent protein gene and neomycin gene were used as positive screening markers and red fluorescent protein gene as negative screening marker.After G418 screening, the resistant clones expressing only green fluorescent protein were selected and used for cross-border PCR detection.The aim of this study was to target porcine myostatin gene by homologous recombination with CRISPR/Cas9.At the target sites of T1 and T2, the positive rates of cells screened by "double fluorescence" strategy were 80.5% and 86.7%, respectively, and the screening efficiency was improved significantly.The "double fluorescence" visual screening strategy developed in this paper has high efficiency and broad spectrum, and has a broad application prospect in animal gene editing field.
【作者单位】：长江大学动物科学学院;湖北省农业科学院畜牧兽医研究所动物胚胎工程及分子育种湖北省重点实验室;湖北三峡职业技术学院农学院;
【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
【分类号】：Q78

【相似文献】