Stable cell line - clone vs. population - (Feb/13/2008 )

Hi guys, I'm trying to transfect PC12 cells with a gene with selection under G418. I've been reading some protocols except I'm confused whether I should use a clone of transfected cells or a population of transfected cells. Any advice would be much appreciated. Thanks very much.

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Generally you would do the transfection in a petri dish, remove the medium, and add G418 medium. Only the transfected cells should survive and you would then isolate individual clone colonies using cloning cylinders and trypsinizing them. I've only done this a few times myself, though, so there may be more efficient ways to do it. Oh and I used FuGene 6 from Roche. It is an easy protocol to follow.

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A single stable clone would be better for experiments than a population. just my way suggestion. In fact depends on the experiment.

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for my experiments, i'm looking at cleavage of my protein. is this case, it is more important to look at a single stable clone or a population of cells? thanks.

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If you choose a single stable clone, you will have the protien being expressed at a particular level. IF you have a population, the cells will be expressing at different levels.

To study the cleavage of a protein, different levels of expression might influence the rate of cleavage and degradation. I guess, it might be better to look at a single clone. Lower level of expression might result in low rate of cleavage. Or if you only wanted to look at cleavage product, then a population might be fine.

You need to think about what are the future plans you want to do with this experiment and then go for it.

good luck !!!

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take a stable transfected clone that reflects the properties of a stable transfected pool

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i don't have much experience in working with cell culture but does one absolutely have to work with clones or would a population be ok also. i am trying to look at the end products following cleavage. i don't even think my lab has the right equipment to isolate clones.

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To pick single clones, you need a 10ul pipette tip. After transfection, you seed the cells on a 10cm plate in a very low density with selection media. After a week or so, you will find individual colonies. You need to pick it up with the pipette tip and then transfer to another cell culture dish and keep in selecting media for some more time. And this would be your single clone.

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i've read about "cloning cylinders". would i have to get one of these because my cells are adherent.

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what are 'cloning cylinders"?

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