Enhancing atrial‐specific gene expression using a calsequestrin cis‐regulatory module 4 with a sarcolipin promoter

Abstract

Background Cardiac gene therapy using the adeno‐associated virus serotype 9 vector is widely used because of its efficient transduction. However, the promoters used to drive expression often cause off‐target localization. To overcome this, studies have applied cardiac‐specific promoters, although expression is debilitated compared to that of ubiquitous promoters. To address these issues in the context of atrial‐specific gene expression, an enhancer calsequestrin cis‐ regulatory module 4 ( CRM4 ) and the highly atrial‐specific promoter sarcolipin were combined to enhance expression and minimize off tissue expression. Methods To observe expression and bio‐distribution, constructs were generated using two different reporter genes: luciferase and enhanced green fluorescent protein (EGFP). The ubiquitous cytomegalovirus (CMV), sarcolipin (SLN) and CRM4 combined with sarcolipin ( CRM4 .SLN) were compared and analyzed using the luciferase assay, western blotting, a quantitative polymerase chain reaction and fluorescence imaging. Results The CMV promoter containing vectors showed the strongest expression in vitro and in vivo . However, the module SLN combination showed enhanced atrial expression and a minimized off‐target effect even when compared with the individual SLN promoter. Conclusions For gene therapy involving atrial gene transfer, the CRM4 .SLN combination is a promising alternative to the use of the CMV promoter. CRM4 .SLN had significant atrial expression and minimized extra‐atrial expression.

Insulin-stimulated endoproteolytic TUG cleavage links energy expenditure with glucose uptake

“…A plasmid encoding HA-tagged ubiquitin was a gift of Dr. Edward Yeh (Addgene #18712) 80 . A plasmid containing the sarcolipin promotor driving luciferase (pTR.SLN.Luc) was a gift of Dr. Dongtak Jeong 81 .…”

“…A pTR-SLN-Luc plasmid, containing 1029 nt from the human SLN promoter upstream of firefly luciferase, was a gift of Dr. Dongtak Jeong 81 . The pRL-SV40P plasmid, containing the SV40 promoter driving Renilla luciferase, was a control and was a gift of Ron Prywes (Addgene plasmid #27163) 88 .…”

The matricellular protein CCN5 prevents adverse atrial structural and electrical remodelling

“…Generation of AAV expressing CCN5, AAV9‐CCN5, was described in our previous work 7 . AAV9‐VLP (Virus‐like particle), viral particles that contain no viral genome, 17 was used as a control virus throughout this study.…”

Hepatitis B Virus X Protein-Induced RORγ Expression to Promote the Migration and Proliferation of Hepatocellular Carcinoma

“…The luciferase reporter system of ROR γ was conducted as follows. The promoter sequence of ROR γ was obtained for GeneCopoeia (Rockville, MD, USA), a web portal for promoter region clones (http://www.genecopoeia.com) [24]. The detailed ROR γ promoter sequence in this study contains the core promoter sequence of ROR γ , which was reported previously [8].…”