A transfection method for short interfering RNA with the lipid-like self-assembling nanotube, A6K
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ORIGINAL PAPER
A transfection method for short interfering RNA with the lipid-like self-assembling nanotube, A6K Daizo Yoshida • K. Kim • I. Takumi • F. Yamaguchi • K. Adachi • A. Teramoto
Received: 8 December 2011 / Accepted: 19 April 2012 / Published online: 5 February 2013 Ó The Japanese Society for Clinical Molecular Morphology 2013
Abstract The aim of the present study was to develop a novel transfection method for short interfering RNA (siRNA). A nanotube with surfactant activity, A6K, consisting of six alanine residues and a hydrophilic head, lysine, was compared to the conventional cationic transfectant reagents siFECTOR and Lipofectamine 2000. Cytotoxicity for the human glioblastoma cell lines U87MG, A172, and T98G was examined with the MTS assay. Transfection efficiency was analyzed with FITC-labeled siRNA targeting matrix metalloproteinase (MMP)-2 mRNA by fluorescent activity on microscopy. The ultrastructure of A6K was evaluated by electron microscopy. The level of cytotoxicity associated with A6K in the U87MG cells was significantly lower than with siFECTOR and Lipofectamine 2000. Transfection efficiency for siRNA was increased in a dose- and time-dependent fashion. The relative expression of MMP-2 mRNA to b-actin was reduced in a dose-dependent manner by real-time RT-PCR analysis. The ultrastructure of the A6K was transformed to micelle formation when mixed with the siRNA. The lipid-like self-assembling peptide, A6K, has genes in the micelle associated with the hydrophilic tail. This transfection method is a novel and stable technique with lower cytotoxicity than the current standard methods. Keywords siRNA
Gene delivery Lipid-like nanotubule
D. Yoshida (&) K. Kim I. Takumi F. Yamaguchi K. Adachi A. Teramoto Department of Neurosurgery, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan e-mail: [email protected]
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Introduction Gene delivery is the process by which foreign genes are introduced into host cells. Gene delivery is, for example, one of the steps necessary for gene therapy as well as the genetic modification of crops. Many different methods of gene delivery have been developed for various types of cells and tissues, from bacterial to mammalian species [1]. Generally, the methods can be divided into two categories, viral and nonviral. Virus-mediated gene delivery utilizes the ability of a virus to inject its DNA into a host cell; a gene that is intended for delivery by this method is packaged into a viral particle. Nonviral methods include physical methods such as microinjection, use of a gene gun, hydrostatic pressure, electroporation, and continuous infusion, as well as sonication and chemical methods such as lipofection [2]. The use of polymeric gene carriers is another method of gene transfer [3]. Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane; they are composed of a phospholipid bilayer [4]. This lipid-based tr
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