Abstract
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.
Highlights
Similar to retro−/lenti- virus system, DNA transposons are useful tools for stable expression of exogenous genes in mammalian cells
We developed a series of vectors with various gene expression cassettes flanked by Sleeping Beauty (SB) inverted repeats, inverted repeat-direct repeat left/right regions (IRDR-L/R), which is recognized by the SB transposase, providing a great convenience tool for molecular cell biology experiments: a) CAG promoter was employed for high expression; b) Gateway design was combined with the vectors to make constructions more convenient; c) vectors with various tags, Flag, HA, GFP, etc., offer more options for different purposes; d) A system expresses N-terminally triple-tagged SFB (S-protein, Flag, and streptavidin-binding peptide) peptides for tandem affinity purification; e) SB delivered CRISPR-Cas9 system was created to achieve virus-free gene knockout
Using tandem affinity purification (TAP) purification system combined with mass spectrometry, we identified NFATc1 as a potential substrate of FBW7 (Fig. 2e and f)
Summary
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. SB-derived vector system for proteins purifying/tracking and gene knockout are still not available. Engineered gene expression is a basic technique in molecular and cellular biology investigations. Vectors containing exogenous genes can be transfected into mammalian cells by chemical transfection or electroporation. It takes long time to get stable expression of exogenous genes in cell lines using virus-free integrating vector, such as pcDNA3 series of vectors. Retro−/lenti- virus systems are the most popular options. The utility of retro−/lenti- viral vectors is heavily restricted by the size of genes.
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