Hello, for my experiment I'm working with SKOV-3-Luc cells, which are SKOV-3 cells stably/continuously expressing the firefly luciferase enzyme. These cells are obtained by the transfection of SKOV-3 cells with a recombinant luciferase expression vector (pGL3-Basic plasmid) with an inserted human NF-kappa B promoter driving luciferase expression (see: Article RNAi-mediated gene-targeting through systemic application of...
).But I'm wondering what the maximum passage number of these cells (SKOV-3 cells and SKOV-3-Luc cells) is? Does the stable expression of the firefly luciferase enzyme has an influence on the maximum passage number of the cells?
And also, how do these kind of modulations/modifications (the induced, stable expression of luciferase enzyme) affect the characteristics of the SKOV-3 cells?
SKOV3 is an immortal cancer cell. There is no maximum passage.
You can expect the luciferase construct to decrease expression over very long culture periods due to genetic drift. You can often slow that down by selecting, and can bring it back by subcloning.
It is not possible to be certain how quickly loss of the construct might happen. It might well be different each time you try, due to the random nature of the process.
Luciferase expression by itself ought not to affect the characteristics of the cells. But the clonal selections in the stable cell line development process might. Again, you can't predict this, and it's going to be different in every clone. Define what characteristics you care about, and develop a test for them. Then, test different clones until you get one that reliably does what you need.
John Schloendorn thank you for your answer. I think what you described is happening now. The cells are losing their construct, but how can I select the cells that still have the transgene (firefly luciferase enzyme, luc+ gene, pGL3 plasmid)?
Hi Lisa,
Looks like there isn't actually a selective marker on that plasmid? In that case it will be very difficult to make stable cell lines. If you want to find rare cells that integrate the plasmid into their genome, and inherit it to their daughter cells, you'll need to use a plasmid with a resistance cassette like puromycin or G418. After that, pick single colonies and test them separately.
- Badges
- Science topic