川崎医科大学 教員情報   
     


  クリバヤシ フトシ   Futoshi Kuribayashi
  栗林 太
   所属   川崎医科大学  医学部 基礎医学 生化学
   職種   教授
論文種別 原著
単著/共著の別 共著
言語種別 英語
査読の有無 査読あり
表題 A dual reporter splicing assay using HaloTag-containing proteins
掲載誌名 正式名:Current Chemical Genomics
巻・号・頁 6,27-37頁
著者・共著者 Koichi Oshim, Takahiro Nagase, Kohsuke Imai, Shigeaki Nonoyama, Megumi Obara, Tomoyuki Mizukami, Hiroyuki Nunoi, Hirokazu Kanegane, Futoshi Kuribayashi, Shin Amemiya, Osamu Ohara
発行年月 2012/09
概要 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.