OBJECTIVE
To create a stable-transfectant that expresses NF155 constitutively
MATERIALS
– pCMV6-NF155-myc-DDK purchased from Origene.
– Geneticin/G418 Life Technologies
– Chicken polyclonal anti-NF
– GAC Alexa 488
– Lipofectamine
PROCEDURE
– Transfect NF155 vector using conventional protocol in a 150 mm dish
– The day after add G418 2mg/mL to the medium. Abundant cell death is seen the day after.
– 2 days later change medium to a G418 concentration of 1mg/mL
– Let the cells regrow and pass them several passes to select only those that have incorporated the transgene. Change medium with G418 1mg/mL every two days.
– Test the ability of the stable transfectant to make the NF155 protein by ICC after 3 weeks of passes (regular ICC, performed as usual)
– Considering that they look good we proceed with cell sorting after 1 month of transfection.
– Prepare 6 150mm plates with the stable line. 3 hours before sorting trypsinize the cells, wash 2xPBS-BSA 2% and add anti-NF (chicken) 1/1000, 30′ on ice. Wash 3xPBS-BSA and add GAC Alexa 488 1/500 30′ on ice. Wash 3xPBSA-BSA and sort.
– For sorting we chose the 5% greenest population. See results.
– Obtained 600.000 cells to plate in a 60mm dish with G418 0.5mg/mL medium
RESULTS
CONCLUSIONS
– After plating the cells they look healthy and start proliferating slowly.
– Future ICC will confirm purity.
