Imaging target mRNA and siRNA-mediated gene silencing in vivo with ribozyme-based reporters.
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.
So
Min-Kyung
MK
Molecular Imaging Program at Stanford, Department of Radiology, Biophysics, Cancer Biology Programs, Stanford University School of Medicine, 1201 Welch Road, Stanford, California 94305-5484, USA.
Gowrishankar
Gayatri
G
Hasegawa
Sumitaka
S
Chung
June-Key
JK
Rao
Jianghong
J
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D005091:Exons; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics*; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D014176:Protein Biosynthesis; D020871:RNA Stability; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000201:enzymology / Q000235:genetics; D013997:Time Factors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D016337:RNA, Catalytic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D008156:Luciferases; D001618:beta-Lactamases