多转座子载体的构建及转座特性比较研究

doi:10.3864/j.issn.0578-1752.2015.11.018

摘要/Abstract

摘要： 【目的】转座子（transposon, Tn）是染色体上可自主复制和移位的基本单位，普遍存在生物体基因组内，Sleeping beauty（SB）、piggyBac（PB）和Tol2分别来源于鲑鱼、甘蓝尺蠖蛾和青鳉鱼，是目前脊椎动物中活性较高的转座子。比较了这3种转座子在哺乳动物细胞的转染、插入和剪切效率，从而获得细胞水平上的高活性转座子系统。【方法】通过高保真PCR法分别从SB、PB和Tol2 3种转座子载体克隆3种转座子3′和5′端的转座元件，测序正确后，将各转座元件依次亚克隆至pT2-HB载体框架，构建成包含这3种转座子的多转座子载体pT3-PST。将绿色荧光蛋白表达框CAG-GFP和新霉素表达框PGK-NEO分别克隆至pT3-PST载体，获得两个表达载体pT3-PST-CAG-GFP和pT3-PGK-NEO。将这两个表达载体分别和转座酶表达质粒pCMV-SB100X、 pCMV-HAhyPBase、pCMV-Tol2以1﹕1质量比混合，经多聚阳离子PEI包裹，共转染小鼠胚胎成纤维细胞（3T3），同时以突变失活的转座酶质粒SB△DD与转座子载体共转染作为阴性对照组。转染GFP 36 h后，用荧光显微镜进行检测GFP表达率，提取细胞基因组，以Amp基因作为内参，进行实时荧光定量PCR，检测GFP插入拷贝数；根据被剪切位置上下游序列设计引物，进行实时荧光定量PCR，检测3种转座子的剪切效率。细胞转染NEO 48 h后，用浓度为500 µg·mL^-1的G418进行耐药性筛选，至正常细胞基本全部死亡（10 d），将细胞进行吉姆萨染液染色，统计耐药细胞数，从而比较各组转座活性。【结果】成功构建了多转座子载体PT3-PST、pT3-PST-CAG-GFP和pT3-PGK-NEO。实验组转染细胞效率均大于50%，且差异不显著（P＞0.05）。SB组GFP相对拷贝数高于Tol2和PB组，但差异不显著（P＞0.05），均显著高于对照组（P＜0.05）。SB和PB组剪切效率显著高于Tol2组（P＜0.05），但SB和PB组之间差异不显著（P＞0.05）。pT3-PGK-NEO与转座酶共转染3T3细胞，G418抗性筛选结果表明，SB组耐药细胞数显著高于PB和Tol2组（P＜0.05），但PB和Tol2两组差异不显著（P＞0.05），此3组均极显著高于阴性对照组（P＜0.01）。【结论】SB转座子系统在细胞水平的转座效率优于PB和Tol2，为细胞水平的转基因研究提供有效基因转移工具。

关键词: SB转座子, PB转座子, Tol2转座子, 胚胎成纤维细胞, 转座活性

Abstract: 【Objective】 Transposons are the basic unit of chromosomes which can autonomously replicate and shift within the whole genome. Sleeping beauty (SB), piggyBac(PB) and Tol2, found from salmonid, cabbage looper moth Trichoplusia ni and Oryzias latipes, respectively, are most active transposons in the vertebrates today. This paper compares the transfection, insertion and cutting efficiencies of the 3 different transposons in 3T3 cells, then obtained the best transposon system at the cellular level.【Method】 The 3′ and 5′ terminal transposable elements were cloned from SB, PB and Tol2 transposon vectors using the high-fidelity PCR in this experiment, ensured the accuracy through sequencing, then the transposable elements were subcloned one-by-one into the pT2-HB carrier frame, thus constructed the multi-transposon vectors, pT3-PST, which included all 3 transposons. Green fluorescent protein expression cassette (pCAG-GFP) and neomycin expression cassette (NEO) genes were cloned into pT3-PST carrier, resulting in two expression vectors, pT3-PST-CAG-GFP and pT3-PGK-NEO. These two expression vectors, with the transposase expression plasmid pCMV-SB100X, were mixed with pCMV-HAhyPBase and pCMV-Tol2 at a mass ratio of 1:1, respectively. They were then wrapped by polycation PEI, and co-transfected into the mouse embrynic fibrilasts (3T3). At the same time, a negative control group was set up using the inactive transposase vector SB△DD. After 36 h post-transfection of GFP, detection of GFP expression was done by fluorescence microscopy, and real-time fluorescent quantitative PCR, after cell genome extraction and using Amp as an internal reference. Primers were designed according to the upstream and downstream sequences, then the cutting efficiency of the 3 transposons was determined by real-time fluorescent quantitative PCR. After 48 h post-transfection of NEO, cells were filtered by using 500 µg·mL^-1 G418 resistance screening until the normal cells were almost dead (10 d). The cells were stained with Giemsa stain, and the number of drug-resistant cells was counted, and finally different transposon activities of different groups were compared.【Result】Multi-transposon vectors, PT3-PST, pT3-PST-CAG-GFP,and pT3-PGK-NEO were successfully constructed. The transfection efficiency of cells in the experimental groups was more than 50%, and the difference was not significant (P＞0.05). The GFP relative copy number of SB group was higher than Tol2 and PB groups, but the difference was not significant (P＞0.05), though it was significantly higher than the control group (P＜0.05). The cutting efficiency of SB group and PB groups was significantly higher than Tol2 group(P＜0.05), though SB and PB group had no significant difference (P＞0.05). pT3-PGK-NEO and transposase co-transfected 3T3 cells, and the results of G418 selection showed that the number of drug-resistant cells of SB group was significantly higher than PB and Tol2 groups (P＜0.05), but the difference between PB and Tol2 groups was not significant (P＞0.05). However, all the three experiment groups were significantly higher than that of the control group (P＜0.01).【Conclusion】The SB transposon system efficiency, at the cellular level, was better than PB and Tol2, which could provide an effective tool for cellular transgenetic research.

Key words: Sleeping beauty, piggyBac, Tol2, embryo fibroblast, transposon activity

沈丹，谢雨琇，李庆平，薛松磊，史云强，王赛赛，陈才，钱跃，高波，崔恒宓，宋成义. 多转座子载体的构建及转座特性比较研究[J]. 中国农业科学, 2015, 48(11): 2270-2278.

SHEN Dan, XIE Yu-xiu, LI Qing-ping, XUE Song-lei, SHI Yun-qiang, WANG Sai-sai, CHEN Cai, QIAN Yue, GAO Bo, CUI Heng-mi, SONG Cheng-yi. Construction of Multi-Transposon Vectors, and Comparative Study of Transposon Characteristics[J]. Scientia Agricultura Sinica, 2015, 48(11): 2270-2278.

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