一种由piggyBac转座子介导高效构建稳定细胞株的策略  

Strategy of efficient construction of stable cell lines mediated by piggyBac transposon

作　　者：盛哲津[1] 王亮 周斌[3] 李利妹 SHENG Zhejin;WANG Liang;ZHOU Bin;LI Limei(School of Life Sciences and Technology,Tongji University,Shanghai 200092,China;Shanghai Pudong New Area Hospital of Traditional Chinese Medicine,Shanghai 200120,China;Department of Vascular Surgery,East Hospital,Tongji University School of Medicine,Shanghai 200120,China;Research Center for Translational Medicine,East Hospital,Tongji University School of Medicine,Shanghai 200120,China)

机构地区：[1]同济大学生命科学与技术学院,上海200092 [2]上海市浦东新区中医医院,上海200120 [3]同济大学附属东方医院血管外科,上海200120 [4]同济大学附属东方医院医学转化平台,上海200120

出　　处：《实验技术与管理》2021年第3期45-50,共6页Experimental Technology and Management

基　　金：中央高校基本科研业务费专项资金资助项目(1507219059);同济大学第15期实验教改项目(2000104092)。

摘　　要：构建稳定细胞株的关键是如何提高外源目的基因插入基因组的效率。piggyBac转座子(简称PB)通过"切离-粘贴"的机制实现在基因组上的转座,其本质是在基因组的不同位点间进行插入。利用PB转座子高效插入基因组的特性,并通过改造该转座系统,即分别在PB转座酶的3’端和5’端加入核定位信号肽,然后在人源胚胎肾293细胞系中评价改造后的PB转座子介导获得稳定细胞株的能力。结果表明,野生型PBase组的克隆形成率是对照组的6.7倍,3’NLSPBase组比野生型PBase组再提高1.3倍。利用人宫颈癌HeLa细胞进一步验证上述结果。可见,经改造后的PB转座子能够提高外源目的基因插入基因组的效率,实现高效构建稳定细胞株。Insertion of foreign target genes into the genome is crucial to the construction of stable cell lines. piggyBac transposon(PB) can translocate between different loci in the genome via the "Cut-and-paste" mechanism. The nature of this process is the insertion of foreign target genes into different sites in the genome. A nuclear localization signal peptide is added to the 3’ or 5’ ends of PB transposase respectively to modify the PB transposon system. The efficiency of the construction of stable cell lines mediated by PB transposon is evaluated in the human embryonic kidney 293 cell line. The results demonstrate that the clone formation rate of wild-type PBase group is 6.7 times that of the control group, and that of 3’NLSPBase group is 1.3 times higher than that of wild-type PBase group. The above results are verified in HeLa cells. The modified PB transposon can improve the efficiency of exogenous target gene insertion into the genome, which will help to achieve high efficient construction of stable cell lines.

关 键 词：PIGGYBAC转座子 稳定细胞株 基因组整合 

分 类 号：Q2[生物学—细胞生物学]