Are all cell lines suitable for transfection to establish a stable expression? I wanted to stably express a gene in a human neuroblastoma cell line. Any suggestions?
To know the yield of transfection the best manner is to transfect a plasmid coding for a GFP protein is to measure the number of positive cells by cytometry (including the viability).
Hi,
you need to transfect the cells with GFP construct with different ratios of your transfection reagent. say lipofectamine in the ration of 1:1, 1:2 etc in a multi-well plate and observe the fluorescence intensity as well as cell viability.
At one point you might see lot of signal but not too many cells. So basically a balance between no. of cells transfected and the signal intensity needs to be seen and that ratio can be used for transfection for that particular cell line.
Trying different transfection reagent may also be a good idea. And transfection to be done in a media without antibiotics.
The cell lines dont like... but you can try it with different transfection reagent as says Shreyas.
You're asking about STABLE transfection, right?
I only read posts about transient expression above, so here are some comments on stable cell line generation:
If you want to do this with a (linearized) plasmid, you first need to find a protocol for transient transfection that works on your cell line. as others mentioned, e.g. a pCMV-GFP and Lipofectamine/Fugene/TransIT or similar are the way to go.
Being transfectable transiently and stably is a property of each cell line and is not predictable, so you have to try different reagents or methods like electroporation. There are databases with already optimized protocols for popular cell lines that make this easier (see each company's homepage for their respective reagent or electroporator).
If transient transfection works, stable transfection will work as well in most cases. Genomic integration of a plasmid is a rare stochastic event (usually something like 1 in 10000 to 1 in 10 million cells, depends on cell line), so you will have to transfect a lot of cells, select them for around two or three weeks and analyze a lot of single cell clones for expression (will be very heterogeneous depending on copy number and integration site). Your Plasmid should have a selectable marker like G-418 or blasticidin resistance.
If you have to do this for more than one gene and more than one cell line, you will have it much easier with lentiviral or retroviral transduction. All people I know working with neuronal cells use lentiviruses.
I agree with Zillinger.
For stable selection one needs to have a selection marker in the plasmid. Try with a reporter system like EGFP first Find out the optimal conditions in which you get maximum transfection. Once that is achieved, you can use your plasmid, which I hope has a selection marker. Add your drug, whatever survives is your stable cell line. But you might want to check this over a period of time.
If plasmid tranfection does not work get a viral transduction done. I reckon viral integration will work.
- Badges
- Science method