The in vivo minigene approach to analyze tissue-specific splicing

• Published in: Brain research. Brain research protocols 1999-12, Vol.4 (3), p.383-394
• Main Authors: Stoss, Oliver, Stoilov, Peter, Hartmann, Annette M., Nayler, Oliver, Stamm, Stefan
• Format: Article
• Language: English
• Subjects: Alternative splicing; Alternative Splicing - genetics; Animals; Cells, Cultured; Cloning, Molecular - methods; Exons; Genes, Synthetic - genetics; Genetic Complementation Test; Humans; Introns; Kidney - cytology; Minigene; Mutagenesis; RNA Precursors - genetics; RT-PCR; Transfection; Transfection - methods
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(99)00043-4

The exact mechanisms leading to alternative splice site selection are still poorly understood. However, recently cotransfection studies in eukaryotic cells were successfully used to decipher contributions of RNA elements ( cis-factors), their interacting protein components ( trans-factors) or the cell type to alternative pre-mRNA splicing. Splice factors often work in a concentration dependent manner, resulting in a gradual change of alternative splicing patterns of a minigene when the amount of a trans-acting protein is increased by cotransfections. Here, we give a detailed description of this technique that allows analysis of large gene fragments (up to 10–12 kb) under in vivo condition. Furthermore, we provide a summary of 44 genes currently investigated to demonstrate the general feasibility of this technique. Themes: Cellular and molecular biology Topics: Gene structure and function: general