title: A Dual Reporter Splicing Assay Using HaloTag-containing Proteins.
abstract: 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.
authors.affiliation: Department of Human Genome Research, Kazusa DNA Research Institute, Kisarazu, Japan ; Laboratory for Immunogenomics, Research Center for Allergy and Immunology, RIKEN, Yokohama Institute, Yokohama, Japan ; Department of Pediatrics, Saitama Medical University, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Oshima
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nagase
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Imai
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Nonoyama
authors.forename: Megumi
authors.initials: M
authors.lastname: Obara
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Mizukami
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nunoi
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kanegane
authors.forename: Futoshi
authors.initials: F
authors.lastname: Kuribayashi
authors.forename: Shin
authors.initials: S
authors.lastname: Amemiya
authors.forename: Osamu
authors.initials: O
authors.lastname: Ohara
keywords: CYBB gene; Chronic granulomatous disease; HaloTag fusion protein; genetic mutation.; luciferase reporter assay; splicing