High-performance gene expression and knockout tools using sleeping beauty transposon system

BackgroundSimilar 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.ResultsIn 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.ConclusionOur 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.

背景 与逆转录病毒/慢病毒系统类似，DNA转座子是在哺乳动物细胞中外源基因稳定表达的有用工具。在近期研究中，睡美人（SB）转座子已被用于将基因整合到宿主基因组中。然而，用于蛋白质纯化/追踪以及基因敲除的基于SB的载体系统仍然不可用。 结果 在本研究中，我们构建了一系列包含睡美人反向重复序列（IRDR - L/R）的载体（称为pSB载体），这些载体可被SB转座酶转座。将Gateway盒连接到pSB载体上以方便克隆。构建了带有各种标签（Flag、Myc、HA、V5和SFB）的载体，以供多种选择。此外，我们将CRISPR - Cas9盒整合到pSB质粒中用于基因敲除。实际上，利用其中一种载体（pSB - SFB - GFP），我们进行了串联亲和纯化，并确定NFATc1是FBW7的一种新型结合伴侣。我们还分别使用pSB - CRISPR载体敲除了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系列载体是在哺乳动物细胞中进行基因过表达和敲除的便捷且强大的工具，为分子细胞生物学研究提供了一种新的替代方法。