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Related Experiment Video

Updated: May 17, 2026

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Published on: September 15, 2021

A Dual Reporter Splicing Assay Using HaloTag-containing Proteins.

Koichi Oshima¹, Takahiro Nagase, Kohsuke Imai

¹Department 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.

Current Chemical Genomics

|November 9, 2012

Summary

This summary is machine-generated.

We developed a dual reporter minigene assay to study mRNA splicing. This method accurately assesses splicing efficiency and identified a mutation affecting CYBB gene splicing in chronic granulomatous disease.

Keywords:

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

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Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

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Published on: April 26, 2017

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

mRNA splicing is a critical post-transcriptional process.
Genetic variations can disrupt normal mRNA splicing, leading to disease.
Accurate assessment of splicing efficiency is crucial for understanding genetic disorders.

Purpose of the Study:

To develop and validate a novel minigene-based splicing assay.
To quantitatively evaluate mRNA splicing efficiency using dual reporters.
To investigate the impact of a specific mutation on CYBB gene splicing.

Main Methods:

Developed a dual reporter minigene assay using luciferase and HaloTag.
Monitored splicing events via luciferase activity and HaloTag protein analysis.
Utilized SDS-PAGE for quantitative insights into mRNA species.
Applied the assay to study a CYBB gene mutation in chronic granulomatous disease.

Main Results:

The dual reporter minigene assay provides reliable estimates of splicing efficiency.
Confirmed the assay's ability to detect both correctly and aberrantly spliced mRNA.
Identified a G>C mutation at the 5'-splice donor site of CYBB intron 5.
Demonstrated that this mutation alters the splicing balance of introns 4, 5, and 6.

Conclusions:

The developed minigene assay is effective for studying mRNA splicing.
This assay can quantify splicing efficiency and detect splicing defects.
The identified CYBB mutation disrupts mRNA splicing, contributing to chronic granulomatous disease.
The assay provides valuable insights into genotype-phenotype correlations in genetic diseases.