Creating stable cell lines - (Oct/28/2008 )
How to create stable cell lines to examine the function of a protein?
If I have primary lung alveolar type II cells (passage 2) or A549 lung adenocarcinoma cells (passage 62), which do I use? Passage 62 is high I think, but the other cells are primary?
How do I get started with this?
Thanks a million!
A549 is already a stable cell line, it has been in culture since the 1970's I think (yep, 1972 according to ATCC).
Some things to consider are:
What effect does long term culture have on protein expression (for your protein of interest)?
What is the native expression of your protein like in each cell line?
Do you need to force your cell to express this protein? If so what effect will this have on the cells?
Primary cell lines have a Hayflick limit (look it up, you are in the homework section after all), how will this affect what you want to do?
How are cancer cell lines different to non-cancer cell lines?
Some things to consider are:
What effect does long term culture have on protein expression (for your protein of interest)?
What is the native expression of your protein like in each cell line?
Do you need to force your cell to express this protein? If so what effect will this have on the cells?
Primary cell lines have a Hayflick limit (look it up, you are in the homework section after all), how will this affect what you want to do?
How are cancer cell lines different to non-cancer cell lines?
Thanks for your response. Our prof never mentioned Hayflick limit. I will have to look that up!
Basically what we have to figure out what to do is this: "produce stable cell lines to examine the function of 'BRILLIANT'" ("BRILLIANT" is just a code word for a particular protein for which I just have a partial sequence.) Also, design experiements to assess functional roles of the protein. And-explain in detail alternative approaches to generating stable cell lines.
Out prof is awful! She's never taught before and has no idea how to do it.. so now were are suffereing. She expects us to know how to do things she hasn't taught us. And get this.. we don't have a book for the class!!!!
Do you a good reference for creating stable cell lines that I could look up?
Thanks!
Try R.I. Freshney, Culture of Animal Cells for a good cell culture reference. I can't remember if it talks about creating stable transfected cell lines or not, but it does discuss a lot of the problems with cell culture.
The usual way of making a cell line that expresses a gene definitely is to couple the gene to a resistance gene for a eukaryotic antibiotic on a plasmid, transfect the cells and then select using the antibiotic. Just the same as doing bacteria selection. There are other methods, that may be all or one of: site specific for DNA insertion, single copy insertion, and inducible. Invitrogen makes a variety of transfection products and a few kits for making expression cell lines. FLP-In T-REx is one system, though it is quite complicated compared to many systems.
Primary cells can be transformed (made immortal) by a variety of processes - if you have a cell type that expresses "brilliant" then you could take some of those cells into culture and then transform them (usually done with a virus in the lab as this speeds up the process somewhat).
Note: cancer cells are all transformed, that is the reason they can divide and grow without senescing/dying, sometimes this is caused by a virus, sometimes not.
Awesome, thanks! I finally got it all figured out! I immortalized the primary cells with hTERT, then went on with Tet-Off. I learned a lot from that manual!