转录激活因子样效应物敲除klf4 b基因的质粒构建与鉴定  

Construction and Identification of TALENs Plasmid Targeting klf4 b Gene

作　　者：毛强[1,2] 邱超[1] 鲁黎[1,3] 马海燕[3] 程彬[1] 梁丹[2] 罗大极[1,4] 

机构地区：[1]武汉大学基础医学院,武汉市430071 [2]通城县人民医院,湖北省咸宁市437400 [3]荆门市第一人民医院,湖北省荆门市448000 [4]发育源性疾病湖北省重点实验室,武汉市430071

出　　处：《医学分子生物学杂志》2015年第3期125-131,共7页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30900853）,教育部高等学校博士点专项研究基金新教师计划（No.20090141120015）.

摘　　要：目的： KLF4是哺乳动物细胞中调控和维持全能性的关键因子，构建与鉴定klf4 b基因敲除的有效位点与相关质粒，以期构建klf4 b基因敲除动物模型研究其功能。方法转录激活因子样效应物核酸酶是目前最有效敲除和编辑基因的基因工程技术之一，设计和组装了klf4 b基因TALENs质粒，在胚胎和整体动物水平验证其有效性。结果成功组装了两对klf4 b基因TALENs质粒，显微注射胚胎后，经检测其中一组成功诱导klf4 b靶向区域突变，突变类型既有插入型也有缺失型；遗传检测显示这些突变型高效的遗传给了下一代。结论在斑马鱼中有效敲除了klf4 b基因，成功获得了斑马鱼klf4 b突变群体；为进一步阐释斑马鱼中klf4 b基因的生物功能提供了良好的研究材料，也为全面理解哺乳类klf4基因的功能提供了研究模型。Objective Kruppel-like factor 4 （ KLF4 ） is a key factor that can regulate and maintain the pluripotency of cells in mammals-The effective sites and related plasmids of klf4 bknockout were constructed and identified in an attempt to establish the animal models of klf4 bknockout and examine theklf4 bgene function-Methods Transcription activator-like effector nucle-ases （ TALENs ） are one of the tools for effective gene knockout and editing at present-Klf4 bTALENs were designed and assembled and their effectiveness was verified at embryonic and animal levels-Results Two pairs of klf4 bTALENs were successfully designed and assembled-One pair of klf4 b-2TALEN could successfully induce the mutation of klf4 bgene in zebrafish, and both insertion and deletion mutations were identified in zebrafish embryos after microinjection of klf4 b-2-TALEN mRNA-These mutations could be efficiently transmited to the next generation-ConclusionKlf4 bgene was effectively knocked out to obtain the klf4 b mutant zebrafish, which provides the good study models for better understanding the biological function of klf4in mammals.

关 键 词：klf4基因 基因敲除 转录激活因子样效应物 斑马鱼 

分 类 号：Q341[生物学—遗传学]