The combination of a synthetic promoter and a CMV promoter improves foreign gene expression efficiency in myocytes.
Skeletal muscle is becoming an attractive target tissue for gene therapy. Nevertheless, the low level of gene therapeutic expression in this tissue is the major limitation to it becoming an ideal target for gene transfer. The promoter is important element for gene transcription; however, the gene expression efficiencies and specificities of viral promoters and skeletal muscle-specific promotors are in themselves limiting factors. In this study, we established a dual-promoters system in skeletal muscle using a cytomegalovirus (CMV) promoter and a skeletal muscle-specific synthetic promoter. Mouse myoblast cell line C2C12 cells were transfected with the system. We demonstrated that the dual-promoters system could significantly improve exogenous gene expression rate in vitro when compared with a single CMV promoter system and a skeletal muscle-specific synthetic promoter system in C2C12 cell line, by 69.48% and 41.93%, respectively. Next, we evaluated the system efficiency in vivo, the results showed that the dual-promoters system increased gene expression in mice 1.23-fold and 1.60-fold, respectively compared with expression controlled by the two single promoter vectors. Finally, we tested the dual-promoters system in growth hormone-releasing hormone (GHRH) gene therapy, and revealed that when these two promoters co-drove the GHRH gene expression in vivo animal growth was enhanced significantly. All these results indicate that use of the dual-promoter vector was more efficient for gene expression in skeletal muscle tissue than use of the single promoter vectors. These finding could, hopefully, lead to the development of a high efficiency expression system in myocytes and form an ideal approach for gene therapy.
Department of Biochemistry, Guangzhou Medical College, Guangzhou 510182, PR China.
Dai
D
Jianwei
Zhang
Z
Qianqian
Liu
L
Songcai
Zhang
Z
Mingjun
Ren
R
Xiaohui
Hao
H
Linlin
Jiang
J
Qingyan
Zhang
Z
Yongliang
D000818:Animals; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D013007:Growth Hormone-Releasing Hormone / Q000096:biosynthesis* / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*
D013007:Growth Hormone-Releasing Hormone