Abstract
Although several bioinformatic tools exist to predict the effect on splicing of a nucleotide change, experimental verification with minigenes is essential for diagnostic purposes, as well as for revealing disease mechanisms and monitoring therapeutic interventions. Minigenes are splice reporter vectors (also known as exon-trapping vectors) that allow confirmation of the effect of mutations on the splicing process, indicated when patients’ samples for RNA studies are not available. The minigene vector codes for exonic portions of a gene defined by functional 5′ splice donor and 3′ splice acceptor sites separated by intronic sequences where a polylinker is located. Here, the exon carrying the mutation under study is cloned along with its flanking intronic sequence. The resulting construct, in its wild-type and mutant sequence version, is transfected in established cell lines and the vector splicing pattern is analyzed. Ideally, the wild-type minigene results in correct exon inclusion, while the mutant construct results in exon skipping or other aberrant transcripts.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Lopez-Bigas N, Audit B, Ouzounis C, Parra G, Guigo R (2005) Are splicing mutations the most frequent cause of hereditary disease? FEBS Lett 579:1900–1903
Ward AJ, Cooper TA (2009) The pathobiology of splicing. J Pathol 220:152–163
Dhir A, Buratti E (2010) Alternative splicing: role of pseudoexons in human disease and potential therapeutic strategies. FEBS J 277:841–855
Baralle D, Baralle M (2005) Splicing in action: assessing disease causing sequence changes. J Med Genet 42:737–748
Spurdle AB, Couch FJ, Hogervorst FB, Radice P, Sinilnikova OM (2008) Prediction and assessment of splicing alterations: implications for clinical testing. Hum Mutat 29:1304–1313
Baralle D, Lucassen A, Buratti E (2009) Missed threads. The impact of pre-mRNA splicing defects on clinical practice. EMBO Rep 10:810–816
Pinotti M, Rizzotto L, Balestra D, Lewandowska MA, Cavallari N, Marchetti G, Bernardi F, Pagani F (2008) U1-snRNA-mediated rescue of mRNA processing in severe factor VII deficiency. Blood 111:2681–2684
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, CSH, NY
Freshney RI (2005) Culture of animal cells. A basic technique, 4th edn. Wiley-Liss, Toronto
Vega AI, Perez-Cerda C, Desviat LR, Matthijs G, Ugarte M, Perez B (2009) Functional analysis of three splicing mutations identified in the PMM2 gene: toward a new therapy for congenital disorder of glycosylation type Ia. Hum Mutat 30:795–803
Abuhatzira L, Makedonski K, Galil YP, Gak E, Zeev BB, Razin A, Shemer R (2005) Splicing mutation associated with Rett syndrome and an experimental approach for genetic diagnosis. Hum Genet 118:91–98
Rincon A, Aguado C, Desviat LR, Sanchez-Alcudia R, Ugarte M, Perez B (2007) Propionic and methylmalonic acidemia: antisense therapeutics for intronic variations causing aberrantly spliced messenger RNA. Am J Hum Genet 81:1262–1270
Baralle M, Skoko N, Knezevich A, De Conti L, Motti D, Bhuvanagiri M, Baralle D, Buratti E, Baralle FE (2006) NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region. FEBS Lett 580:4449–4456
Buratti E, Baralle M, Baralle FE (2006) Defective splicing, disease and therapy: searching for master checkpoints in exon definition. Nucleic Acids Res 34:3494–3510
Acknowledgements
This work was supported by grant SAF2007-61350 from Comisión Interministerial de Ciencia y Tecnología. The authors also acknowledge the support received from Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and the institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Desviat, L.R., Pérez, B., Ugarte, M. (2012). Minigenes to Confirm Exon Skipping Mutations. In: Aartsma-Rus, A. (eds) Exon Skipping. Methods in Molecular Biology, vol 867. Humana Press. https://doi.org/10.1007/978-1-61779-767-5_3
Download citation
DOI: https://doi.org/10.1007/978-1-61779-767-5_3
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-766-8
Online ISBN: 978-1-61779-767-5
eBook Packages: Springer Protocols