Design of a GFP reporter for splicing analysis in mammalian cells

Q1 Immunology and Microbiology

Biotechnology Reports Pub Date : 2025-03-13 DOI:10.1016/j.btre.2025.e00887

Arthur T. Menezes, Helder Y. Nagasse, Hilan R. M. Lopes, Patricia P. Coltri

{"title":"Design of a GFP reporter for splicing analysis in mammalian cells","authors":"Arthur T. Menezes, Helder Y. Nagasse, Hilan R. M. Lopes, Patricia P. Coltri","doi":"10.1016/j.btre.2025.e00887","DOIUrl":null,"url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"46 ","pages":"Article e00887"},"PeriodicalIF":0.0000,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Reports","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2215017X25000141","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Immunology and Microbiology","Score":null,"Total":0}

引用次数: 0

Abstract

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.

查看原文本刊更多论文

用于哺乳动物细胞剪接分析的GFP报告基因的设计

真核生物基因由外显子和内含子组成。Pre-mRNA剪接促进外显子连接和内含子去除，由一种称为剪接体的特殊大分子机制完成，剪接体由五个小核糖核蛋白颗粒（snRNPs）和一百多种蛋白质组成。这种配合物的活性是高度准确的，由于其组分的协调活动。剪接的改变与几种疾病的发展有关，包括神经退行性疾病，如肌萎缩性侧索硬化症和不同类型的癌症。利用剪接报告系统可以详细了解真核细胞中的剪接调节。我们设计了一种适合于哺乳动物细胞剪接分析的报告质粒。我们的报告是基于GFP的表达，拼接结果可以很容易地在荧光显微镜下看到。我们量化了两种人类细胞系HEK-293T和MDA-MB-231的剪接活性，证实了其在活细胞培养中的适用性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Biotechnology Reports Immunology and Microbiology-Applied Microbiology and Biotechnology

CiteScore

15.80

自引率

0.00%

发文量

审稿时长

55 days

期刊介绍： Biotechnology Reports covers all aspects of Biotechnology particularly those reports that are useful and informative and that will be of value to other researchers in related fields. Biotechnology Reports loves ground breaking science, but will also accept good science that can be of use to the biotechnology community. The journal maintains a high quality peer review where submissions are considered on the basis of scientific validity and technical quality. Acceptable paper types are research articles (short or full communications), methods, mini-reviews, and commentaries in the following areas: Healthcare and pharmaceutical biotechnology Agricultural and food biotechnology Environmental biotechnology Molecular biology, cell and tissue engineering and synthetic biology Industrial biotechnology, biofuels and bioenergy Nanobiotechnology Bioinformatics & systems biology New processes and products in biotechnology, bioprocess engineering.