摘要
为研究睡美人(Sleeping Beauty,SB)转座子系统在草鱼(Ctenopharyngodon idellus)肾脏细胞(CIK)中介导的整合特性,构建了SB转座子和转座酶在两个质粒的二元反式(trans)转座子系统,以及转座子和转座酶元件在同一个质粒的一元顺式(cis)转座子系统;通过转染CIK细胞,用荧光显微镜、流式细胞仪和荧光定量PCR分析了转染2d后及嘌呤霉素筛选4周后的细胞,测定Ds Red转染效率和整合效率,并结合高效热不对称交互式PCR扩增获得SB转座子整合位点的序列。结果表明,SB二元转座子系统的整合效率远高于一元系统;SB转座子与转座酶比例为1鲶2时,外源基因Ds Red的整合效率最高;SB转座子偏向于插入草鱼基因组TA序列处。研究表明优化SB转座子和转座酶的比例能提高外源基因在草鱼细胞中的整合效率并快速获得突变细胞,同时为在鱼类细胞中采用SB转座子建立突变体文库提供理论基础。
In this study,the features of SB-mediated integration were examined in grass carp(Ctenopharyngodon idellus)kidney cells(CIK).Two expression systems were constructed:the transposase and transposon reporters were separatedinto two vectors designated as SB trans-configuration(trans)system,whereas the transposase and transposon cassetteswere combined in one vector named as SB cis-configuration(cis)system.The vectors were transfected into CIKcells,and then the transfection and integration efficiency of DsRed were investigated by fluorescence microscope,flowcytometry,and quantitative RT-PCR after2days or4weeks of puromycin selection.In addition,the unknown sequenceswithin the integration regions were also characterized by hiTAIL-PCR.The results showed that the integrationefficiency of SB trans system was much higher than the cis system;the highest integration efficiency of DsRed was observedwhen the ratio of transposon versus transposase was1:2;the transposon was inserted randomly into TA dinucleotideswith preference.In conclusion,this study demonstrated that optimizing the ratio of SB transposon versus transposaseincreased the exogenous gene integration efficiency and generated mutant cells rapidly.
作者
陈凯
薛亭
王艺舟
潘启华
于淼
陈天圣
CHEN Kai;XUE Ting;WANG Yi-Zhou;PAN Qi-Hua;YU Miao;CHEN Tian-Sheng(Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, College of Fisheries, Huazhong AgriculturalUniversity, Wuhan 430070, China;College of Fishery, Engineering Technology Research Center of Henan Provincefor Aquatic Animal Cultivation, Henan Normal University, Xinxiang 453007, China)
出处
《水生生物学报》
CAS
CSCD
北大核心
2018年第2期264-270,共7页
Acta Hydrobiologica Sinica
基金
国家重点基础研究计划 (2013CB967700)
国家自然科学基金(31672653和31771648)
华中农业大学自主科技创新基金(2013RC014和2662015PY049)资助
