Abstract: 【Abstract】 Objective To search for the most suitable gene transfection conditions for rat muscle in vivo by therapertic ultrasound-mediated microbubble destruction (UMMD). Methods A mixture of microbubbles and enhanced green flurescence protein (EGFP) plasmids was injected into rat tibialis anterior muscle and the muscels treated with ultrasound irradiation by different output intensity, duty cycle and irradiation time of therapeutic ultrasound. The transfection efficiency was demonstrated by the EGFP expression results under fluorescent staining and immunohistochemical staining. And the most favorable ultrasound conditions as well as local intra-muscle and intravenous injection methods were selected based on the best transfection efficiency and the least muscle damage under HE staining. The rats were divided into four groups: ① ultrasound + microbubbles + plasmid; ② microbubble + plasmid; ③ ultrasound + plasmid; ④ plasmid only. The favorable ultrasound conditions and injection method were selected out on the basis of above steps. EGFP expression was observed in the tibialis anterior muscle of each group. The rats were sacrificed in groups at 5 days after they underwent ultrasound irradiation, the EGFP expression in muscle was observed after fluorescent and immunohistochemical stainingand the muscle damage was also observed under HE staining.Results The most favorable conditions consisted of a 1 MHz therapeutic ultrasound irradiation applied for 3 min, a power output of 2 W/cm2 and a 20% duty cycle. Under these conditions, the muscle showed significant EGFP expression, and the muscle was not significantly damaged. The EGFP expression induced by the local intra muscle injection was more significantly increased than that induced by the intravenous injection.Among the four groups, the EGFP expression under fluorescence staining and immunohistochemical staining in the muscle of the ultrasound + microbubbles + plasmid group was significantly higher than that of the other three groups, and the microbubbles + plasmid group was higer than that of the other two groups (P<0.05). No muscle damage caused by the ultrasound and microbubbles was detected under HE staining. Conclusion Under the optimal transfection conditions, the therapeutic ultrasound mediated microbubble destruction method can significantly enhance the in vivo gene transfection efficiency of rat muscle and found no damage to the muscle. Thus, these conditions can be used as part of a safe and effective non viral gene transfection procedure for gene therapy.