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baoxiniao金虫 (小有名气)
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[求助]
In vivo siRNA transfection
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Transfection solution was prepared as described previously (33). In brief, 10 l of each of the 10 mol/l siRNA stock solutions was mixed with 330 l of sterile 0.9% NaCl solution, containing 8.3% transfection reagent (TKO; Mirus) 10 min before the transfection procedure. Total concentration of siRNAs in the final transfection solution was 0.8 M. Nonrelated siRNA directed against green fluorescence protein (eGFP; Euroffins MWG, Denmark) was used as control for side effects of the transfection procedure (sham transfection). In experiments addressing expression of contractile phenotype marker gene expression, a sham-transfection procedure using 0.9% NaCl solution without siRNA and transfection reagent was used as additional control. Rats were anesthetized by a subcutaneous injection of combination of hypnorm (3 mg/100 g; VetaPharma, Leeds, UK) and midazolam (1.5 mg/100 g; Hameln Pharm, Langes Feld, Germany). Every 30 min one third of the initial dose of the anesthetic was supplied by a subcutaneous injection. siRNA transfection was performed as described previously (33). Shortly, a medial laparotomy was performedand a section of mesentery was extracted and spread on a sterile cloth, dampened with sterile salt solution (0.9% NaCl). A segment of first order branch of mesenteric artery (1 to 2 mm) was gently cleaned, and a microfil cannula (World Precision Instruments) filled with the transfection solution was inserted into the artery. The transfection solution was injected into the lumen and kept there for 20 min, while downstream flow was occluded with a surgical clamp. The clamp was released and the arterial branches were flushed with a new portion of the transfection solution, each 2 to 3 min. Care was taken to avoid overdistension of the arterial segment since this was seen to induce unrelated changes in protein expression in the arterial wall (unpublished observations). The operation field was covered by cloth dampened with a sterile salt solution throughout the procedure. Body temperature was maintained at 37.5°C by a thermostatically controlled heating platform. Painkiller (0.2 ml/kg; Temgesic, Schering-Plough Europe, Belgium) was injected subcutaneously at the end of operation. Rats were euthanized by CO2-inhalation at 3 days or, where indicated, 10 days after operation, and mesenteric arteries were used for further studies. We have previously shown that this transfection procedure leads to uptake of siRNA into the VSMCs. |
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