基于Cas9技术的无标记定点整合LacS基因的载体系统构建  

Construction of unmarked fixed-point integration Lac gene vector system based on the Cas9 technology

作　　者：刘晓[1] 苏小虎[1] 马云龙[1] 郭梓茹 张焱茹[1] 周欢敏[1] 张立[1] LIU Xiao, SU Xiaohu, MA Yunlong, GUO Ziru, ZHANG Yanru, ZHOU Huanmin, ZHANG Li(Life Science College of Inner Mongolia Agricuhure University, Hohhot 010018, Chin)

机构地区：[1]内蒙古农业大学生命科学学院

出　　处：《黑龙江畜牧兽医》2018年第7期75-78,248,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31460599)

摘　　要：为了获得奶牛Cas9特异识别位点载体,同时构建无标记Lac S基因载体,并对Cas9载体进行活性验证,以达到建立Lac S基因阳性细胞系的目的,试验基于奶牛β-casein第2内含子区域序列设计Cas9靶位点作为潜在Lac S基因插入位点、构建Cas9/gRNA载体,利用T7E1酶进行活性验证,构建包含针对靶位点微同源序列的Lac S基因载体。结果表明：经过PCR和测序比对,发现构建的Cas9/gRNA载体具有活性,而且Lac S基因载体可用于整合。同时构建的包含有微同源序列和Cas9靶位点的Lac S基因克隆载体,可用于后续研究。The aim of the present study was to obtain the vector of the cow Cas9 specific recognition sit and the vector of unmarked Lac S gene.Thus not only the enzyme activity of the vector could be identified but also Lac S gene positive cell lines could be established. The Cas9/gRNA vector was constructed by using the second intron regional sequence of the cow beta-casein as the Cas9 target location and potential Lac S gene insertion site. Then the enzyme activity of the vector was identified by using the T7E1 enzyme. Meanwhile,a Lac S gene vector containing the micro-homologous sequence of target site was constructed. The results showed that the Cas9/gRNA vector had enzyme activity and the Lac S gene vector could be used for integration by PCR and sequencing. And the Las gene cloning vector containing the micro-homologous sequence and the Cas9 target point were constructed,which laid the foundation for the follow-up study.

关 键 词：CRISPR/Cas9 LacS基因载体 无标记 微同源 奶牛 

分 类 号：Q782[生物学—分子生物学]