Hi,
I have been transducing MDA-MB-468 and MV-3 cells with lentivirus, however not succesfully.
The transfection: we used a polymer-based transfection method (PEI).
The transduction was performed by adding viral particles to cancer cells for 24 hours, then replacing the supernatant with full medium (with serum and Pen/Strep) for another 24 hour. The selection was performed with Blasticidin for 4 days. The plasmid contains a resistance gene for Blasticidin, so the transduces cells should survive.
After selection all cells died, the ones that were transduced but also the ones that were not transduced and serve as negative control.
To me it seems like either (1) the transduction did not work or (2) the selection procedure is too harsh. However, i tried increasing the transduction incubation from 1 day to 3 days, an i tried using lower and higher Blasticidin concentrations. Both did not work for me.
have you tried to transduce your lentivirus to easy cells (such as 293) to confirm the gene expression by western blot?
do you have a plate centrifuge, spin for 1 hour will enhance the transduction.
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