title: Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing.
abstract: All human genes contain a diverse array of cis-acting elements within introns and exons that are required for correct and efficient precursor messenger RNA (pre-mRNA) splicing. Recent computational analyses predict that most human exons contain elements required for splicing coinciding with an appreciation for the high frequency with which mutations that disruption pre-mRNA splicing cause disease. Minigenes provide a means to directly determine whether disease-causing mutations or single nucleotide polymorphisms (SNPs) affect splicing efficiency. Minigenes have also been instrumental in investigations of alternative splicing to identify cis elements required for cell-specific splicing events, demonstrating regulation of individual splicing events by specific RNA binding proteins, and correlating binding of these splicing regulators with splicing regulation. Here we present a versatile minigene plasmid vector designed for rapid cloning and analysis of cis elements and trans-acting factors that influence splicing efficiency or regulate cell-specific splicing. Ubiquitous expression and unique restriction sites allow for straightforward replacement of a variety of gene segments to analyze the effects of nucleotide substitutions on splicing, to identify tissue-specific regulatory elements, or to determine responsiveness to coexpressed proteins or small molecules.
authors.affiliation: Baylor College of Medicine, Houston, TX, USA.
authors.forename: Gopal
authors.initials: G
authors.lastname: Singh
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Cooper
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation; D002460:Cell Line; D002522:Chlorocebus aethiops; D005091:Exons; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009154:Mutation; D032446:Myoblasts / Q000502:physiology; D010957:Plasmids; D012322:RNA Precursors / Q000502:physiology*; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D015534:Trans-Activators / Q000502:physiology*
chemical_list: D012322:RNA Precursors; D015534:Trans-Activators