A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Abstract

Summary: Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off‐target effects the therapeutic gene should be driven by a tissue‐specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue‐specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle‐specific expression but presents heat‐shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b‐deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle‐specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431–438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

Loss of zebrafish Smyd1a interferes with myofibrillar integrity without triggering the misfolded myosin response

“…In fact, similar expression pattern of smyd1a was also observed for other teleost fish such as the Chinese Perch [15]. The transgenic expression of a Smyd1a-GFP fusion protein in zebrafish embryos using the established skeletal and cardiac muscle tissue specific unc45b promoter [11] revealed a clear sarcomeric pattern in striated muscle tissue, which alternates with a-Actinin, suggesting Smyd1a localizing to the sarcomeric M-band in vivo similar to Smyd1b. The recruitment of Smyd1b to the M-line was shown to be highly dependent on Phe223 and Ser225 within its sequence [16].…”

“…As reported before, expression of Smyd1b-GFP in the zebrafish line Tg(unc45b min :-smyd1b-GFP) revealed a striated pattern, which alternates with the Z-disc marker a-Actinin, demonstrating localization of Smyd1b to sarcomeric M-bands (Fig. 1E) [3], [11]. Interestingly, a clear alternating sarcomeric localization with a-Actinin was also visible for Smyd1a in our transgenic line Tg(unc45b min :smyd1b-GFP) at 72 hpf ( Fig.…”

“…1D). To further analyze subcellular localization of Smyd1a, we generated a transgenic line expressing smyd1a-gfp under control of the unc45b min promoter that drives expression specifically in skeletal and cardiac muscle tissue (Tg(unc45b min :smyd1a-GFP)) [11]. As reported before, expression of Smyd1b-GFP in the zebrafish line Tg(unc45b min :-smyd1b-GFP) revealed a striated pattern, which alternates with the Z-disc marker a-Actinin, demonstrating localization of Smyd1b to sarcomeric M-bands (Fig.…”

Loss of the novel Vcp (valosin containing protein) interactor Washc4 interferes with autophagy-mediated proteostasis in striated muscle and leads to myopathy in vivo

“…The final vector construct for microinjection into zebrafish oocytes and the generation of germline transgenic lines includes the zebrafish striated muscle-specific minimal unc45b promoter and mCherry-EGFP-LC3B (rat LC3B) sequences to visualize autophagy function in striated muscle cells in vivo [42]. …”

Muscle-Specific Promoters for Gene Therapy

“…The unc45b gene encoding the muscle-specific myosin chaperone in fish was also used to develop a muscle-specific promoter [ 72 ]. Thus, the 195-bp promoter fragment (-505...-310) was able to induce expression in skeletal and cardiac muscles in fish and ensured reporter protein expression in mouse muscles when the plasmids were delivered by electroporation.…”