Converse Andrea D, Belur Lalitha R, Gori Jennifer L, Liu Geyi, Amaya Felipe, Aguilar-Cordova Estuardo, Hackett Perry B, McIvor R Scott
Beckman Center for Transposon Research, Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall 321 Church Street S.E., Minneapolis, MN, 55455, USA.
Biosci Rep. 2004 Dec;24(6):577-94. doi: 10.1007/s10540-005-2793-9.
Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.
睡美人（SB）是一种从硬骨鱼中发现的转座子序列重建而来的基因插入系统，能够介导DNA序列从转染质粒转座到脊椎动物细胞群体的染色体中。SB系统由一个转座子和一个转座酶来源组成，转座子由一个两侧带有转座子反向重复序列的目的基因构成。在这里，我们进行了一系列研究，以进一步将SB介导的转座表征为一种将基因转移到染色体并最终用于人类基因治疗的工具。用含有新霉素磷酸转移酶（NEO）基因的转座子转染小鼠3T3细胞、HeLa细胞和人A549肺癌细胞，当与表达SB转座酶的质粒共转染时，耐药集落形成增加了几倍。还发现，当与编码SB转座酶的质粒共转染时，含有抗甲氨蝶呤二氢叶酸还原酶基因的转座子能提高甲氨蝶呤耐药集落形成的频率。一个含有单纯疱疹病毒胸苷激酶基因的质粒以及一个含有NEO基因的转座子被用于在含有G418加更昔洛韦的培养基中对随机重组体（NEO+TK+）进行反选择。有效的反选择需要在转染后有5天的恢复期，然后再转入含有更昔洛韦的培养基中，以便让未稳定转染的细胞中瞬时表达的胸苷激酶活性消退。对克隆分离株的Southern分析表明，当在含有更昔洛韦以及G418 的培养基中分离克隆时，从随机重组事件向转座事件发生了转变。我们发现，在同一质粒上同时包含转座子和转座酶功能，可显著提高Huh7人肝癌细胞中的稳定基因转移频率。这些实验结果为哺乳动物细胞中转座过程以及可能适用于基因治疗研究的转座子和转座酶功能的最佳配置提供了技术和概念上的见解。
