Channel: JoVE (Journal of Visualized Experiments) clear
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| 1 | JoVE (Journal of Visualized Experiments) | Paramyxoviruses for Tumor-targeted Immunomodulation | Protocol Preview | 108 | 37.2 | 2:01 | This method can help address key challenges in the field of cancer immunotherapy by facilitating the development of oncologic vectors for targeted immunomodulation. The main advantage of the applied reverse genetic system is its versatility. Therefore, vectors can be adapted to address various research questions and target tumors with diverse immune signatures. The virus propagation steps, especially determining the optimal time point to harvest virus, are difficult to learn from a text protocol because syncytra formation must be assessed visually over time. To begin, design and clone recombinant immunomodulatory vectors as described in the manuscript. This protocol describes specific steps for the development of an oncologic measles vaccine vector, encoding a bi-specific T cell enclosure. To rescue measles virus from CDNA, 24 hours before transfection, plate measles virus producer cells evenly on a six-wall plate. Seed 200,000 cells in 2 ml DMEM containing 10% FBS per well to achieve 65 to 75% confluency at the time of transfection. Mixed 5 micrograms of recombinant DNA encoding the measles virus anti-genome that will be used to transfect the cells, appropriate plasmids, and a fluorescent reporter in a total volume of 200 microliters of DMEM. At 18.6 microliters of liposomal transfection reagent to the mixture, and immediately flick the tube to mix. Incubate this transfection mix for 25 minutes at room temperature to transfect the MB. | ↗ |