Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import
Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import
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DOI：
10.1038/mt.2009.127
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发表时间：
2009-11-01
影响因子：
12.4
通讯作者：
Dean, David A.
中科院分区：
文献类型：
--
作者：
Miller, Aaron M.;Munkonge, Felix M.;Dean, David A.
虽然转染在实验室中经常使用，但外源DNA运输到细胞核的机制尚不清楚。通过提高我们对载体如何绕过基因转移的众多细胞屏障的理解，可以设计出更有效的基因传递方法。我们已经开始设计质粒结构，在没有细胞分裂的情况下进入特定细胞类型的细胞核，从而提高表达水平。我们已经证明，在质粒结构中包含特定的DNA序列介导了体外和体内的核输入。在这里，我们使用质粒亲和层析、质谱（MS）和转染质粒结构的活细胞下拉来鉴定与这些DNA核靶向序列（dts）以序列特异性方式相互作用的蛋白质辅因子。输入蛋白β(1)、输入蛋白7和小鸟苷三磷酸酶Ran在质粒和拉下实验中都显示出dts特异性相互作用，与我们的质粒核输入模型一致。此外，用小干扰RNA （siRNA）敲低输入蛋白β(1)可消除质粒核输入，表明它是必要的辅助因子。我们的发现，特异性核细胞蛋白介导质粒核输入，可用于设计更有效的载体基因传递。
Although transfections are routinely used in the laboratory, the mechanism(s) by which exogenous DNA is transported into the nucleus is poorly understood. By improving our understanding of how vectors circumvent the numerous cellular barriers to gene transfer, more efficient gene delivery methods can be devised. We have begun to design plasmid constructs that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing levels of expression. We have shown that inclusion of specific DNA sequences in plasmid constructs mediates nuclear import both in vitro and in vivo. Here, we use plasmid affinity chromatography, mass spectrometry (MS), and live-cell pulldowns of transfected plasmid constructs to identify protein cofactors that interact in a sequence-specific manner with these DNA nuclear targeting sequences (DTSs). Importin beta(1), importin 7, and the small guanosine triphosphatase Ran all demonstrate DTS-specific interaction in both MS and pull-down assays, consistent with our model of plasmid nuclear import. In addition, knockdown of importin beta(1) with small interfering RNA (siRNA) abrogates plasmid nuclear import, indicating that it is a necessary cofactor. Our discovery that specific karyopherins mediate plasmid nuclear import can be used to design more effective vectors for gene delivery.