Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan.
Int J Pharm. 2011 Aug 30;415(1-2):218-20. doi: 10.1016/j.ijpharm.2011.05.051. Epub 2011 May 27.
Heterogeneity of transgene expression is a severe disadvantage in the use of cationic lipid-mediated gene vectors. We previously demonstrated that heterogeneity of the post-nuclear delivery process, as well as intracellular trafficking (i.e. nuclear delivery) is a major determinant in the overall heterogeneity in gene expression, when plasmid DNA (pDNA) is transfected to HeLa cells using a lipoplex. In this study, we explored the mechanism underlying this heterogeneity in a post-nuclear transport process by the dual imaging of mRNA and its encoded protein (histone H2B-tagged mTFP1; mTFP1-H2B) in a single cell. To establish a highly sensitive imaging system for mRNA, we used fluorescence in situ hybridization (FISH) combined with tyramide signal amplification (TSA) and a semiconductor quantum dot (QD) probe. The mRNA expression and protein production were quantified by counting the total pixel intensity in the region of interest (r.o.i.) surrounding single cells. As a result, the correlation was poor in a scattered plot of mRNA expression versus protein production in individual cells. These findings demonstrate that cell-to-cell differences in the translation process are also a key factor in heterogeneous gene expression.
转基因表达的异质性是阳离子脂质体介导的基因载体应用的一个严重缺陷。我们之前的研究表明，在使用脂质体转染 HeLa 细胞时，核后传递过程的异质性以及细胞内运输（即核传递）是导致基因表达总体异质性的主要决定因素。在本研究中，我们通过对单个细胞中转录后核运输过程中 mRNA 及其编码蛋白（组蛋白 H2B 标记的 mTFP1；mTFP1-H2B）的双重成像，探讨了这种异质性的机制。为了建立一个用于 mRNA 的高灵敏度成像系统，我们使用了荧光原位杂交（FISH）结合酪胺信号放大（TSA）和半导体量子点（QD）探针。通过对单个细胞周围感兴趣区域（r.o.i.）的总像素强度进行计数，对 mRNA 表达和蛋白产量进行定量。结果，在单个细胞中 mRNA 表达与蛋白产量的散点图上相关性较差。这些发现表明，翻译过程中的细胞间差异也是异质性基因表达的关键因素。
