Gene transfer and expression in human neutrophils.: The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes
Gene transfer and expression in human neutrophils.: The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes
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DOI：
10.1186/1471-2172-7-28
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发表时间：
2006-12-06
期刊：
影响因子：
3
通讯作者：
Catz, Sergio D.
中科院分区：
文献类型：
--
作者：
Johnson, Jennifer L.;Ellis, Beverly A.;Catz, Sergio D.
背景：中性粒细胞是一种分离后存活率较低的非分裂细胞。因此，外源基因在中性粒细胞中的表达具有挑战性。我们在这里报告基因转染和活性蛋白的表达在人原发性外周中性粒细胞使用核转染。结果：转染2 h后，在人中性粒细胞中实现了外源基因表达。我们表明，中性粒细胞转染核感染是功能性细胞，能够响应可溶性和颗粒性刺激。它们保留了经历包括吞噬作用在内的生理过程的能力。利用这种技术，我们能够证明p47（phox）的phox同源（PX）结构域定位于人中性粒细胞的质膜上。我们还发现RhoB，而不是p47（phox）的PX结构域，被转移到成熟吞噬体的膜上。结论：我们证明了外源蛋白在人中性粒细胞中的cDNA转移和表达与细胞活力是相容的，不再是研究人中性粒细胞中蛋白质功能的限制。
Background: Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection.Results: Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX) domain of p47(phox) localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47(phox), is translocated to the membrane of mature phagosomes.Conclusion: We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.