Futoshi Kuribayashi
   Department     ,
Article types 原著
Language English
Peer review Peer reviewed
Title A dual reporter splicing assay using HaloTag-containing proteins
Journal Formal name:Current Chemical Genomics
Volume, Issue, Page 6,27-37頁
Author and coauthor Koichi Oshim, Takahiro Nagase, Kohsuke Imai, Shigeaki Nonoyama, Megumi Obara, Tomoyuki Mizukami, Hiroyuki Nunoi, Hirokazu Kanegane, Futoshi Kuribayashi, Shin Amemiya, Osamu Ohara
Publication date 2012/09
Summary To evaluate the effects of genetic variations on mRNA splicing, we developed a minigene-based splicing assay using reporter genes encoding luciferase and the multifunctional HaloTag protein. In addition to conventional RT-PCR analysis, splicing events can be monitored in this system using two parameters: luciferase activity and signals derived from HaloTag-containing proteins bound to a fluorescent ligand following SDS-PAGE. The luciferase activity reflects the accumulated amounts of successfully spliced HaloTag-luciferase fusion products, whereas the amounts and sizes of HaloTag-containing proteins provide quantitative insights into precursor, correctly spliced, and aberrantly spliced mRNA species. Preliminary experiments confirmed that the dual reporter minigene assay can provide estimates of overall splicing efficiency based on the levels of protein products. We then used the minigene assay to analyze a case of chronic granulomatous disease that was caused by a G>C mutation at position +5 in the 5'-splice donor site of intron 5 of the CYBB gene. We found that the G>C mutation affected CYBB mRNA splicing by changing a delicate balance of splicing efficiencies of introns 4, 5, and 6.
DOI doi: 10.2174/1875397301206010027.