Design of a GFP reporter for splicing analysis in mammalian cells
Year: 2025
Type: article
Abstract: • Generation of a GFP splicing reporter suitable for transfection in mammalian cells. • Splicing can be visualized using fluorescence microscopy. • The reporter plasmid has similar GFP production in HEK-293T and MDA-MB-231 cell lines. Eukaryotic genes are formed by exons and introns. Pre-mRNA splicing promotes exon ligation and intron removal and is performed by a specialized macromolecular machinery named spliceosome, composed of five small ribonucleoprotein particles (snRNPs) and more than one hundred proteins. The activity of this complex is highly accurate due to the coordinated activity of its components. Altered splicing has been related to the development of several diseases, including neurodegenerative disorders, such as amyotrophic lateral sclerosis, and different types of cancer. Detailed understanding of splicing regulation in eukaryotic cells can be achieved using splicing reporter systems. We designed a reporter plasmid suitable for splicing analysis in cultured mammalian cells. Our reporter is based on GFP expression, and the splicing outcome can be easily visualized by fluorescence microscopy. We quantified splicing activity in two human cell lines, HEK-293T and MDA-MB-231, confirming its suitability for use in live cells in culture.
Source: Biotechnology Reports
Authors: Arthur T. Menezes, Helder Y. Nagasse, Hilan R. M. Lopes, Patricia P. Coltri
Institution: Universidade de São Paulo
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,480
OpenAlex ID: W4408394266