水生生物学报2018，Vol.42Issue(2)：264-270,7.DOI:10.7541/2018.033
睡美人转座子在草鱼CIK细胞中介导的高效基因整合
THE EFFICIENT GENE INTEGRATION OF SLEEPING BEAUTY TRANSPOSON-MEDIATED IN GRASS CARP (CTENOPHARYNGODON IDELLUS) CIK CELLS
摘要
Abstract
In this study,the features of SB-mediated integration were examined in grass carp(Ctenopharyngodon idel-lus)kidney cells(CIK).Two expression systems were constructed:the transposase and transposon reporters were sepa-rated into two vectors designated as SB trans-configuration(trans)system,whereas the transposase and transposon cas-settes were combined in one vector named as SB cis-configuration(cis)system.The vectors were transfected into CIK cells,and then the transfection and integration efficiency of DsRed were investigated by fluorescence microscope,flow cytometry, and quantitative RT-PCR after 2 days or 4 weeks of puromycin selection. In addition, the unknown se-quences within the integration regions were also characterized by hiTAIL-PCR. The results showed that the integration efficiency of SB trans system was much higher than the cis system;the highest integration efficiency of DsRed was ob-served when the ratio of transposon versus transposase was 1:2; the transposon was inserted randomly into TA dinuc-leotides with preference. In conclusion, this study demonstrated that optimizing the ratio of SB transposon versus trans-posase increased the exogenous gene integration efficiency and generated mutant cells rapidly.关键词
睡美人转座子/转座酶/草鱼肾脏细胞/整合位点Key words
Sleeping Beauty transposon/Transposase/CIK cells/Integration sites分类
农业科技引用本文复制引用
陈凯,薛亭,王艺舟,潘启华,于淼,陈天圣..睡美人转座子在草鱼CIK细胞中介导的高效基因整合[J].水生生物学报,2018,42(2):264-270,7.基金项目
国家重点基础研究计划(2013CB967700) （2013CB967700）
国家自然科学基金(31672653和31771648) （31672653和31771648）
华中农业大学自主科技创新基金(2013RC014和2662015PY049)资助[Supported by the State Key Development Program for Basic Research of China(2013CB967700) （2013RC014和2662015PY049）
the National Natural Science Foundation of China(31672653,31771648) （31672653,31771648）
the Fundamental Research Funds for the Central Universities(2013RC014,2662015PY049)] （2013RC014,2662015PY049）
