| Skeletal muscle is an attractive tissue for somatic gene delivery because it is large, has good capacity for protein synthesis, is easily accessible for intramuscular injection, and has the ability to take up plasmid after intramuscular adminstration. Intramuscular injection of exogenous plasmids has been shown to produce low levels of transgene expression in muscle cells that persist for more than 1 year. Muscle can therefore function as a bioreactor for synthesis and secretion of proteins, leading to systemic herapeutic effects. However, one major limitation of the use of muscle as a gene therapy target has been the relatively low levels of expression achieved with current plasmid expression systems.The cytomegalovirus (CMV) enhancer/promoter (E/P) is generally considered to be the most powerful promoter for gene therapy in muscle, but interestingly the presence of multiple CMV enhancers does not improve gene expression further. Many efforts have been made, with some progress, to improve the level of exogenous gene expression in muscle through the development of novel gene expression systems. For example, a synthetic muscle- specific promoter shows more persistent expression than the CMV promoter.In this reasch, we design and synthesis muscle- specific promoter-SP. Six vectors were constructed,which were carrying EGFP or GRF (pCMV-EGFP, pSP-EGFP, pCMV-SP-EGFP, pCMV-GRF, pSP-GRF and pCMV-SP-GRF) driven by CMV promoter, SP promoter and CMV-SP promoters, respectively.Plasmids of pCMV-EGFP, pSP-EGFP, pCMV-SP-EGFP were injected to skeletal muscle of mice to analysis their expression efficacy respectively. The difference of EGFP expressions of the vectors was evaluated by semiquantitative polymerase chain reaction (PCR) at 1, 2, 3 and 4 weeks postinjection. The results showed that the expression efficacy of EGFP by two promoters was higher than the groups of pCMV-EGFP and pSP-EGFP (44.03 %, P<0.01 and 31.11 %, P<0.01); Furthermore, by the fluorescence detection, we gain the more powerful fluorescence signals of the group pCMV-SP-EGFP.Plasmids of pCMV-GRF, pSP-GRF and pCMV-SP-GRF were injected to skeletal muscle of mice. The accumulative weight gain of mice were measured and the concentrations of serum IGF-I and GH were measured by radioimmunoassay(RIA)in every 10 days. The results showed that injection of pCMV-SP-GRF lead to accumulative weight gain was 38.01%(P<0.01), 19.68 %(P<0.05) and 20.30 %(P<0.01) higher than the groups of the saline group, pCMV-GRF and pSP-GRF at 30 days after injection. The data indicated that two promoters co-priming GRF gene in-vivo could enhance animal growth significantly. The serum IGF-I levels were 195.51 %(P<0.01),154.34 %(P<0.01) and 142.92 %(P<0.01) higher than the groups of the saline group, pCMV-GRF and pSP-GRF. These have a correlation with the accumulative weight gain of mice as well(P<0.05). The results showed that plasmids were expressed in-vivo could promote the secretion of GH and improve the serum IGF-I levels. But the serum GH levels of each group did not have significantly difference, it is possible that the secrete cycle of GH is fairly short, so it is hard to analysis the secretion trend of the whole cycle by a point of data.This research indicated that two-promoters CMV and SP eukaryotic expression vector could improve an exogenous gene expression efficency in skeletal tissue of mice. This will provide a beneficial exploring to improve the efficiency of gene expression in skeletal muscle.
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