ZFN,TALEN和CRISPR/Cas9在小鼠Rosa26基因定点整合外源基因的效率比较  

Efficiency comparison of Rosa26-targeted integration of exogenous gene via ZFN,TALEN or CRISPR/Cas9

作　　者：刘小凤[1] 刘蔚 聂宇 丛佩清[1] 刘小红[1] 陈瑶生[1] 何祖勇[1] LIU Xiaofeng;LIU Wei;NIE Yu;CONG Peiqing;LIU Xiaohong;CHEN Yaosheng;HE Zuyong(School of Life Sciences/State Key Laboratory of Biocontrol,Sun Yat-sen University,Guangzhou 510006,China)

机构地区：[1]中山大学生命科学学院/有害生物控制与资源利用国家重点实验室,广东广州510006

出　　处：《中山大学学报（自然科学版）》2020年第2期137-144,共8页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：国家转基因生物新品种培育重大专项项目(2016ZX08006003-006);广东省自然科学基金(2016A030313310)。

摘　　要：哺乳动物黑色素的合成依赖于酪氨酸的氧化作用,而酪氨酸酶(Tyr)是催化酪氨酸氧化反应的关键酶,当外源Tyr基因整合进白毛色小鼠基因组中,会使它获得黑色素合成的能力,表现出与原来不同的毛色表型。为方便、快捷地获得Tyr基因整合的小鼠,构建了一个无启动子的pTyr-2A-DsRed同源重组质粒供体,选择Rosa26的第一个内含子作为外源基因整合的靶位点,设计了切割位点几乎一致的ZFN、TALEN和CRISPR/Cas9系统。通过流式对比分析C 2C 12细胞中红色荧光蛋白DsRed的表达水平,比较了3种基因组编辑工具介导的外源基因定点整合效率,结果发现CRISPR/Cas9的效率最高,在此基础上,利用CRISPR/Cas9将供体整合到小鼠胚胎干细胞中,筛选单细胞克隆进行囊胚腔注射和胚胎移植,获得一只存活的嵌合体小鼠,表现出白毛中夹杂黑毛的表型,表明整合到小鼠Rosa26的Tyr基因可以正常表达。Mammalian melanin synthesis is dependent on the oxidation of tyrosine.Tyrosinase(Tyr)is a key enzyme that catalyzes the oxidation of tyrosine.When exogenous Tyr is integrated into the genome of white coat color mouse,it can render the mouse to obtain the function of melanin synthesis,presenting a different coat color.To rapidly generate the mouse model with Tyr gene integrated,in this study,we constructed a promotorless plasmid donor pTyr-2A-DsRed,which containis the coding sequences of Tyr gene and the red fluorescent reporter(DsRed),and the two flanking homologous arms.We selected the first intron of Rosa26 for targeting integration,and designed ZFN pair,TALEN pairs and CRISPR/Cas9 cutting at almost the same site.Through measuring the intensity of DsRed fluorescence in C 2C 12 cells by flow cytometry,we compared the efficiency of the targeted integration of exogenous DNA mediated by the three different genome editing tools,and found that CRISPR/Cas9 was the most efficient.Therefore,we further integrated the plasmid donor into the genome of mouse embryonic stem(ES)cells,and screening the targeted single cell clones for blastocyst injection and subsequent embryo transfer.Ultimately,a single survived chimeric mouse with plasmid donor integrated was obtained.This mouse presented a white color hair mixed with black color hair phenotype,indicating that the targeted integration of Tyr gene in Rosa26 locus was correctly expressed.

关 键 词：CRISPR/Cas9 TALEN ZFN Rosa26 定点整合 

分 类 号：Q78[生物学—分子生物学]