title: Dual imaging of mRNA and protein production: an investigation of the mechanism of heterogeneity in cationic lipid-mediated transgene expression.
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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan. akita@pharm.hokudai.ac.jp
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Akita
authors.forename: Yuko
authors.initials: Y
authors.lastname: Umetsu
authors.forename: Dai
authors.initials: D
authors.lastname: Kurihara
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Harashima
mesh_terms: D002412:Cations; D015870:Gene Expression; D006367:HeLa Cells; D006657:Histones / Q000235:genetics; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008164:Luminescent Proteins; D057054:Molecular Imaging / Q000379:methods*; D014176:Protein Biosynthesis; D021381:Protein Transport; D011506:Proteins / Q000235:genetics*; D045663:Quantum Dots; D012333:RNA, Messenger / Q000235:genetics*; D014162:Transfection; D019076:Transgenes
chemical_list: D002412:Cations; D006657:Histones; D008055:Lipids; D008164:Luminescent Proteins; D011506:Proteins; D012333:RNA, Messenger