Mann David G J, McKnight Timothy E, McPherson Jackson T, Hoyt Peter R, Melechko Anatoli V, Simpson Michael L, Sayler Gary S
Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, USA.
ACS Nano. 2008 Jan;2(1):69-76. doi: 10.1021/nn700198y.
RNA interference (RNAi) has become a powerful biological tool over the past decade. In this study, a tetracycline-inducible small hairpin RNA (shRNA) vector system was designed for silencing cyan fluorescent protein (CFP) expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% +/- 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene-silencing effects using RNAi in single cells and cell populations.
在过去十年中，RNA干扰（RNAi）已成为一种强大的生物学工具。在本研究中，设计了一种四环素诱导型小发夹RNA（shRNA）载体系统，用于沉默青色荧光蛋白（CFP）的表达，并将其与黄色荧光蛋白（yfp）标记基因一起通过刺入接种到空间索引垂直排列的碳纳米纤维阵列（VACNF）上，导入中国仓鼠卵巢细胞。VACNF结构实现了多个基因的同时递送、界面细胞的后续粘附和增殖以及对单个细胞随时间的重复监测。在刺入接种和四环素诱导后，53.1%±10.4%的刺入接种细胞被诱导型CFP沉默shRNA载体完全沉默。此外，在仍表达CFP的细胞群体中的单个细胞中也观察到了有效的CFP沉默。这种有效的瞬时表达系统能够在单细胞和细胞群体中使用RNAi快速分析基因沉默效应。
