Zebrafish dystrophin and utrophin genes: Dissecting transcriptional expression during embryonic development

Abstract

Abstract. Some genes can encode multiple overlapping transcripts, and this can result in challenges in identifying transcript-specific developmental expression profiles where tools such as RNA in situ hybrisations are inapplicable. Given this difficulty, we have undertaken a preliminary analysis of the developmental expression profile of selected transcripts of the dystrophin and utrophin genes of the zebrafish (Danio rerio) by targeting unique and common regions of each of these transcripts. The dystrophin and utrophin genes of zebrafish were identified by bioinformatic analysis and the dystrophin gene predictions were confirmed by transcript sequencing. These data enabled primer pairs to be designed in order to determine the expression profiles of unique, but overlapping transcripts, throughout embryonic development using quantitative real time reverse transcription PCR (qRT-PCR). The data indicated the early expression of the short carboxyl-terminal dystrophin transcript, with expression of the full length muscle transcript occurring during myogenesis. Importantly, a composite of these two profiles appeared to comprise the major transcriptional load of the zebrafish dystrophin gene. In contrast, utrophin gene expression was dominated by the full length transcript throughout embryogenesis. The approach described here provided a means by which a gene's transcriptional complexity can be deconvoluted to reveal transcriptional diversity during embryogenesis. This approach, however, required the identification of unique regions for transcript-specific targeting, and an appreciation of alternative splicing events that may compromise the design of primers for qRT-PCR.

In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

“…GFP was expressed from pCMV-GFP (Addgene 11153). Full-length zebrafish Dystrophin ( Lai et al, 2012 ) GFP tagged was synthesized by GenBrick and subcloned into pCI-Neo at the MluI-SalI site (GenScript USA Inc., Piscataway Township, NJ, United States). All constructs were fully sequenced.…”

“…Dystrophin is well studied in zebrafish and its homology with the human Dystrophin is well documented ( Guyon et al, 2003 ; Jin et al, 2007 ; Berger et al, 2011 ; Lai et al, 2012 ). Several mutant and transgenic lines have been used as model for Duchenne muscular dystrophy and testing potential therapeutic targets ( Kunkel et al, 2006 ; Johnson et al, 2013 ; Kawahara and Kunkel, 2013 ; Waugh et al, 2014 ; Wood and Currie, 2014 ).…”