Browsing by Author "Escorza Sius, Amanda Paz"

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    Efficient transfection protocol of rainbow trout gills epithelial (RTgill-W1) cell line through nucleofection system. v1
    (2024) Escobar Aguirre, Sebastián Gonzalo; Escorza Sius, Amanda Paz; Escobar Aguirre, Matías
    In this protocol, we optimize the experimental condition to express plasmid DNA in salmonid fish rainbow trout gills epithelial (RTgill-W1), using a nucleofection system. The principle of nucleofection is a combination of electrical parameters that ensures efficient DNA delivery into the nucleus, combined with low toxicity and high cell viability. Using the Neon Transfection System (Invitrogen‱ MPK5000; code: 10431915) and the Invitrogen‱ Neon‱ Transfection System Kit 10 μl (brand: Invitrogen‱ MPK1096; code: 10124334) we were able to express over 60% of GFP in RTgill W 1 line cells. These results were achieved after trying different optimization steps voltage (ranges between 850 - 1600 volts), pulse exposure (times 10 30 ms), and number of pulses, being the condition 1600 volts, 20 ms 1 pulse, and 1 μg μL of plasmid the most effective compared to the other conditions, presenting a higher percentage of GFP expression and lower mortality rate.