NDLI: 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

Content Provider	PubMed Central
Author	Ansseau, Eugénie Domire, Jacqueline S. Wallace, Lindsay M. Eidahl, Jocelyn O. Guckes, Susan M. Giesige, Carlee R. Pyne, Nettie K. Alexandra, Belayew Harper, Scott Q.
Editor	Chadwick, Brian P.
Copyright Year	2015
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 preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.
Related Links	http://dx.doi.org/10.1371/journal.pone.0118813
Starting Page	118813
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	3
Volume Number	10
Language	English
Publisher	Public Library of Science
Publisher Date	2015-03-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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Intronic sequences flanking alternatively spliced exons are conserved between human and mouse.

Intrinsic Epigenetic Regulation of the D4Z4 Macrosatellite Repeat in a Transgenic Mouse Model for FSHD

Asymmetric Bidirectional Transcription from the FSHD-Causing D4Z4 Array Modulates DUX4 Production

Aberrant splicing events caused by insertion of genes of interest into expression vectors

Differential DNA methylation of the D4Z4 repeat in patients with FSHD and asymptomatic carriers

The evolutionary distribution and structural organization of the homeobox-containing repeat D4Z4 indicates a functional role for the ancestral copy in the FSHD region

The Evolutionary Distribution and Structural Organization of the Homeobox-Containing Repeat D4Z4 Indicates a Functional Role for the Ancestral Copy in the FSHD Region

A simple answer for a splicing conundrum.

Splice junctions follow a 205-base ladder.

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

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Intronic sequences flanking alternatively spliced exons are conserved between human and mouse.

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Aberrant splicing events caused by insertion of genes of interest into expression vectors

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A simple answer for a splicing conundrum.

Splice junctions follow a 205-base ladder.

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