A generalizable strategy for imaging pre-mRNA levels in living subjects using spliceosome-mediated RNA trans-splicing.
Molecular imaging of gene expression is currently hindered by the lack of a generalizable platform for probe design. For any gene of interest, a probe that targets protein levels must often be generated empirically. Targeting gene expression at the level of mRNA, however, would allow probes to be built on the basis of sequence information alone. Presented here is a class of generalizable probes that can image pre-mRNA in a sequence-specific manner, using signal amplification and a facile method of delivery.


METHODS
Pre-trans-splicing molecules (PTMs) were engineered to capitalize on the phenomenon of spliceosome-mediated RNA trans-splicing. Using a modular binding domain that confers specificity by base-pair complementarity to the target pre-mRNA, PTMs were designed to target a chimeric target mini gene and trans-splice the Renilla luciferase gene onto the end of the target. PTMs and target genes were transfected in cell culture and assessed by luciferase assay, reverse-transcriptase polymerase chain reaction, Western blot, and rapid analysis of 5' cDNA ends. PTMs and target genes were also assessed in vivo by hydrodynamic delivery in mice.


RESULTS
Efficiency and specificity of the trans-splicing reaction were found to vary depending on the binding domain length and structure. Specific trans-splicing was observed in living animals (P = 0.0862, Kruskal-Wallis test).


CONCLUSIONS
Described here is a model system used to demonstrate the feasibility of spliceosome-mediated RNA trans-splicing for imaging gene expression at the level of pre-mRNA using optical imaging techniques in living animals. The experiments reported here show proof of principle for a generalizable imaging probe against RNA that can amplify signal on detection and be delivered using existing gene delivery methodology.
Walls
Zachary F
ZF
Department of Molecular and Medical Pharmacology, Geffen School of Medicine at UCLA, Los Angeles, California, USA.
Puttaraju
M
M
Temple
Gary F
GF
Gambhir
Sanjiv S
SS
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids; D012322:RNA Precursors / Q000378:metabolism*; D048790:Renilla / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017381:Spliceosomes / Q000502:physiology*; D020040:Trans-Splicing; D014162:Transfection / Q000379:methods
D012322:RNA Precursors; D008156:Luciferases