BACKGROUND Similar to retro-/lenti- virus system, DNA transposons are useful tools for stable expression of exogenous genes in mammalian cells. Sleeping Beauty (SB) transposon has adopted for integrating genes into host genomes in recent studies. However, SB-derived vector system for proteins purifying/tracking and gene knockout are still not available. RESULTS In this study, we generated a series of vectors (termed as pSB vectors) containing Sleeping Beauty IRDR-L/R that can be transposed by SB transposase. Gateway cassette was combined to the pSB vectors to facilitate the cloning. Vectors with various tags, Flag, Myc, HA, V5 and SFB, were generated for multiple options. Moreover, we incorporated the CRISPR-Cas9 cassette into the pSB plasmids for gene knockout. Indeed, using one of these vectors (pSB-SFB-GFP), we performed Tandem Affinity Purification and identified that NFATc1 is a novel binding partner of FBW7. We also knocked out RCC2 and BRD7 using pSB-CRISPR vector respectively, and revealed the novel roles of these two proteins in mitosis. CONCLUSION Our study demonstrated that the pSB series vectors are convenient and powerful tools for gene overexpression and knockout in mammalian cells, providing a new alternative approach for molecular cell biology research.

中文翻译：

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使用睡美人转座子系统的高性能基因表达和敲除工具。

背景与逆转录病毒/慢病毒系统类似，DNA转座子是用于在哺乳动物细胞中稳定表达外源基因的有用工具。在最近的研究中，睡美人 (SB) 转座子已用于将基因整合到宿主基因组中。然而，用于蛋白质纯化/跟踪和基因敲除的 SB 衍生载体系统仍然不可用。结果 在这项研究中，我们生成了一系列包含睡美人 IRDR-L/R 的向量（称为 pSB 向量），可以通过 SB 转座酶进行转座。网关盒与 pSB 载体组合以促进克隆。为多个选项生成了带有各种标签、Flag、Myc、HA、V5 和 SFB 的向量。此外，我们将 CRISPR-Cas9 盒整合到 pSB 质粒中以进行基因敲除。事实上，使用这些载体之一（pSB-SFB-GFP），我们进行了串联亲和纯化，并确定 NFATc1 是 FBW7 的新型结合伙伴。我们还分别使用 pSB-CRISPR 载体敲除 RCC2 和 BRD7，并揭示了这两种蛋白质在有丝分裂中的新作用。结论我们的研究表明，pSB系列载体是哺乳动物细胞基因过表达和敲除的方便而强大的工具，为分子细胞生物学研究提供了一种新的替代方法。