Aberrant splicing in transgenes containing introns, exons, and V5 epitopes: lessons from developing an FSHD mouse model expressing a D4Z4 repeat with flanking genomic sequences

PloS One
Eugénie AnsseauScott Q Harper

Abstract

The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD). This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF) alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV) and strong viral control elements (CMV promoter, SV40 poly A) to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM) contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3' untranslated region (pLAM) harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentia...Continue Reading

References

Jan 1, 1986·Annual Review of Biochemistry·R A PadgettP A Sharp
Oct 1, 1997·Journal of Computational Biology : a Journal of Computational Molecular Cell Biology·M G ReeseD Haussler
Sep 29, 2004·Molecular Therapy : the Journal of the American Society of Gene Therapy·Michael J BlankinshipJeffrey S Chamberlain
Oct 7, 2004·American Journal of Human Genetics·Richard J F L LemmersSilvere M van der Maarel
Apr 7, 2005·Proceedings of the National Academy of Sciences of the United States of America·Scott Q HarperBeverly L Davidson
Sep 24, 2005·Annals of Neurology·Petra G M van OverveldSilvère M van der Maarel
Jun 26, 2007·Neuromuscular Disorders : NMD·Valeria KowaljowAlberto L Rosa
Oct 10, 2007·American Journal of Human Genetics·Richard J L F LemmersSilvere M van der Maarel
Nov 7, 2007·Proceedings of the National Academy of Sciences of the United States of America·Manjusha DixitYi-Wen Chen
May 14, 2008·BioTechniques·Bill Brizzard
Sep 12, 2008·Nucleic Acids Research·Jung-Sik KimTodd Waldman
Apr 3, 2009·Nucleic Acids Research·François-Olivier DesmetChristophe Béroud
Sep 4, 2009·Human Mutation·Jessica C de GreefSilvère M van der Maarel
Aug 21, 2010·Science·Richard J L F LemmersSilvère M van der Maarel
Nov 10, 2010·PLoS Genetics·Lauren SniderDaniel G Miller
May 3, 2011·Hybridoma·Linda N GengStephen J Tapscott
Nov 5, 2011·PloS One·Céline VanderplanckAlexandra Belayew
Apr 18, 2012·Molecular Therapy : the Journal of the American Society of Gene Therapy·Lindsay M WallaceScott Q Harper
Jan 4, 2013·Neurology·Lynn M HartweckMichael Kyba
Jul 4, 2013·Human Molecular Genetics·Gregory J BlockDaniel G Miller
Mar 19, 2014·Molecular and Cellular Biology·Charis L HimedaTakako I Jones

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Citations

Sep 28, 2016·Intractable & Rare Diseases Research·Bo BaoToshifumi Yokota
Dec 7, 2016·Continuum : Lifelong Learning in Neurology·Jeffrey M Statland, Rabi Tawil
Jun 22, 2018·Current Treatment Options in Neurology·Ava Y Lin, Leo H Wang
Mar 18, 2020·Nucleic Acids Research·Lukasz CiszewskiLinda Popplewell
Feb 21, 2019·Scientific Reports·Hiroaki MitsuhashiJeffrey Boone Miller

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Methods Mentioned

BETA
transgenics
restriction
electrophoresis
PCR
targeted mutation
transgenic
transfection
restriction digest

Software Mentioned

Human Splicing Finder
NNSPLICE0

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