Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines
Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines
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DOI：
10.1002/biot.201400821
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发表时间：
2015-04-01
影响因子：
4.7
通讯作者：
Marschalek, Rolf
中科院分区：
文献类型：
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作者：
Kowarz, Eric;Loescher, Denise;Marschalek, Rolf
在哺乳动物细胞中稳定的基因表达是许多体外和体内实验的先决条件。然而，无论是将质粒整合到哺乳动物基因组中，还是使用逆转录/慢病毒系统，都有内在的局限性。转座元件的使用，例如睡美人系统(SB)，绕过了大多数这些缺点(整合位点、大小限制)，并允许快速产生稳定的细胞系。Sb的整合过程是由转座酶催化的，这种基因转移系统的操作简单、快速和安全。在这里，我们报告了我们对现有SB载体系统的改进，并提出了两种新的载体类型，用于任何感兴趣基因的健壮的结构性或诱导性表达。这两种类型都有16种变种，具有不同的选择标记(嘌罗霉素、潮霉素、杀菌素、新霉素)和荧光蛋白表达(GFP、RFP、BFP)，以满足大多数实验要求。利用该系统，可以在中等产量的设置下(三到五天)快速、可靠地从稳定的转基因细胞中产生细胞系。细胞系强健地表达任何感兴趣的基因，无论是结构性的还是受到多西环素的严格调控。这使得许多实验室实验能够以快速和可靠的方式加速数据的生成。
Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (three to five days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner.