Application of the lentiviral MMB technique under flow in isolated mouse aortas

C57BL/6J wild type mice (Charles River, Burlington, MA, USA) were euthanized by cervical dislocation under anaesthesia (Midazolam 5 mg/kg; Medetomidin 0.5 mg/kg; Fentanyl 0.05 mg/kg). Thoracal aortas were isolated and intercostal arteries were cauterized to prevent leakage. Aortas were bilaterally catheterized and mounted above a magnet in a recirculation system constructed for MMB-mediated transduction (see Fig. ​Fig.3e).3e). Aortas (diameter ~1 mm) were perfused with serum-free DMEM at around 7-8 dyn/cm². 200 µl of LV-MMB (corresponding to 9.9*10⁷ VP) were diluted 1:5 in HBSS and were slowly injected into the plastic tube upstream of the perfused aorta. US (120 s, 1 MHz, 2 W/cm², 50% duty cycle) was applied simultaneously at the site of magnetic field application. After further 30 min of perfusion and MF exposure aortas were transferred to a cell culture dish with 5 ml of DMEM supplemented with 20% FCS and left for gene expression for 6 days in a humidified incubator before assaying. Aortic ring sprouting assays were performed as previously described⁴³.