Imaging Target mRNA and siRNA-Mediated Gene Silencing In Vivo with Ribozyme-Based Reporters

• Published in: Chembiochem : a European journal of chemical biology Vol. 9; no. 16; pp. 2682 - 2691
• Main Authors: So, Min-Kyung, Gowrishankar, Gayatri, Hasegawa, Sumitaka, Chung, June-Key, Rao, Jianghong
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 03.11.2008; WILEY‐VCH Verlag
• Subjects: Animals; Base Sequence; beta-Lactamases - genetics; beta-Lactamases - metabolism; Cercopithecus aethiops; COS Cells; Exons; Gene Silencing; Genes, p53 - genetics; Genes, Reporter - genetics; Humans; imaging; Luciferases - genetics; Luciferases - metabolism; Mice; Mutation; Protein Biosynthesis; RNA; RNA reporter; RNA Stability; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; RNA, Messenger - analysis; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; siRNA; Tetrahymena thermophila; Tetrahymena thermophila - enzymology; Tetrahymena thermophila - genetics; Time Factors; trans‐splicing ribozymes
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200800370

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.