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  • Title: Dual imaging of mRNA and protein production: an investigation of the mechanism of heterogeneity in cationic lipid-mediated transgene expression.
    Author: Akita H, Umetsu Y, Kurihara D, Harashima H.
    Journal: Int J Pharm; 2011 Aug 30; 415(1-2):218-20. PubMed ID: 21641986.
    Abstract:
    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.
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