A Dual Reporter Splicing Assay Using HaloTag-containing Proteins

Koichi Oshimaa, b, g, Takahiro Nagasea, Kohsuke Imaic, Shigeaki Nonoyamac, Megumi Obarad, Tomoyuki Mizukamid, Hiroyuki Nunoid, Hirokazu Kaneganee, Futoshi Kuribayashif, Shin Amemiyag, Osamu Ohara*, a, b
a Department of Human Genome Research, Kazusa DNA Research Institute, Kisarazu, Japan
b Laboratory for Immunogenomics, Research Center for Allergy and Immunology, RIKEN, Yokohama Institute, Yokohama, Japan
c Department of Pediatrics, National Defense Medical College, Tokorozawa, Japan
d Department of Pediatrics, Miyazaki Medical College, Miyazaki, Japan
e Department of Pediatrics, Graduate School of Medicine, University of Toyama, Toyama, Japan
f Department of Biochemistry, Kawasaki Medical School, Kurashiki, Japan
g Department of Pediatrics, Saitama Medical University, Japan

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© Oshima et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the Department of Human Genome Research, Kazusa DNA Research Institute, Kisarazu, Chiba Japan; Tel: +81-438-52-3913; Fax: +81-483-52-3914; E-mail:


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.

Keywords:: Chronic granulomatous disease, CYBB gene, HaloTag fusion protein, luciferase reporter assay, splicing, genetic mutation.