ABSTRACT
We previously developed an in vivo site-specific transfection method using a suction device in mice; namely, a tissue suction-mediated transfection method (tissue suction method). The aim of this study was to apply the tissue suction method for cardiac gene transfer. Naked plasmid DNA (pDNA) was intravenously injected in mice, followed by direct suction on the beating heart by using a suction device made of polydimethylsiloxane. We first examined the effects of suction conditions on transgene expression and toxicity. Subsequently, we analyzed transgene-expressing cells and the transfected region of the heart. We found that heart suction induced transgene expression, and that -75 kPa and -90 kPa of suction achieved high transgene expression. In addition, the inner diameter of the suction device was correlated with transgene expression, but the pressure hold time did not change transgene expression. Although the tissue suction method at -75 kPa induced a transient increase in the serum cardiac toxicity markers at 6 h after transfection, these markers returned to normal at 24 h. The cardiac damage was also analyzed through the measurement of hypertrophic gene expression, but no significant differences were found. In addition, the cardiac function monitored by echocardiography remained normal at 11 days after transfection. Immunohistochemical analysis revealed that CD31-positive endothelial cells co-expressed the ZsGreen1-N1 reporter gene. In conclusion, the tissue suction method can achieve an efficient and safe gene transfer to the beating heart in mice.
Subject(s)
Heart/physiology , Myocardium/metabolism , Transfection/methods , Transgenes/genetics , Animals , Creatine Kinase, MB Form/blood , Dimethylpolysiloxanes/chemistry , Echocardiography , Gene Expression , Mice , Myocardium/pathology , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Plasmids/administration & dosage , Plasmids/genetics , Plasmids/metabolism , Pressure , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Transfection/instrumentation , Troponin T/bloodABSTRACT
BACKGROUND: We previously developed a suction-mediated transfection method in mice. PURPOSE: The purpose of this study was to optimize the suction-mediated transfection conditions using a pressure-controlled computer system for efficient and safe kidney-targeted gene delivery in mice. METHODS: Naked pCMV-Luc was injected into the tail vein in mice, and then the right kidney was suctioned by a device of the suction pressure-controlled system. The effects of renal transfection conditions, such as the suction pressure degree, suction pressure waveform and device area were evaluated by measuring luciferase expression. In addition, renal injury was examined. RESULTS: The renal suction-mediated transfection method at -30 kPa showed high transgene expression. The renal suction waveform did not affect the transfection activity. Under the optimized conditions, the high transgene expression was mostly observed at the renal suctioned site. The transfection conditions used did not induce histological defects or increases in two renal injury biomarkers (Kidney injury molecule-1 mRNA and Clusterin mRNA). DISCUSSION AND CONCLUSION: We have clarified the transfection conditions for efficient and safe transfection in the kidney using the suction-mediated transfection method in mice.
Subject(s)
Gene Transfer Techniques , Kidney/metabolism , Suction/methods , Transfection/methods , Animals , Biomarkers/metabolism , Female , Genetic Therapy/methods , Luciferases/metabolism , Mice , Mice, Inbred ICR , Plasmids/metabolism , Pressure , RNA, Messenger/metabolismABSTRACT
We previously developed an in vivo tissue suction-mediated transfection method (denoted as the tissue suction method) for naked nucleic acids, such as plasmid DNA (pDNA) and small interfering RNA (siRNA), in mice. However, it remains unclear whether the suction pressure conditions affect the results of this method. Therefore, in the present study, we assembled a computer system to control the suction pressure and investigate the effects of the suction pressure conditions on the efficiency of the liver suction transfection of naked pDNA that encodes luciferase in mice. Using the developed system, we examined the effects of the minimum magnitude of the suction pressure, suction pressure waveform, and suction times of the luciferase expression level in mice livers. We determined that the liver suction method at 5 kPa was not only effective but also caused the lowest hepatic toxicity in mice. Additionally, the results indicated that the suction pressure waveform affects the luciferase expression levels, and a single period of suction on the targeted portion of the liver is sufficient for transfection. Thus, the developed system is useful for performing the tissue suction method with high accuracy and safety.