Transfect primary cells - is it possible? too difficult? (Jun/06/2005 )

Hello, I will be transfecting (ectopically, transiently) my gene of interest to study its localization/interaction with a certain protein. I was originally going to do this in COS7 or HeLa cells, but my colleague does not want to do that. He insists we use primary cells, because they are not as "screwed up" as transformed cell lines.

1) I just need to see whether my protein interacts with this other known protein. I don't care whether it is in a "screwed up" background. I understand his concern, but I don't think its really necessary in this experiment. I would use a regular mammalian expression vector for this. Who is right? Me or him?

2) How difficult is it to transfect primary cells - say, human skin fibroblasts? Would I have to use some other fancy vector to transfect primary cells? I don't want to use Amaxa's apparatus because I don't want to buy all that stuff. I just need a quick and dirty assay about the protein interactions. If it looks promising then we could design some cool fancy experiments for further study.

Please help. Soon. Thank you very much in advance.

s70048

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hi
transfecting primary cells do not encounter more problem than one cell type (i mean special cares). The major point is that as they're primary cells, you need to be relative quick to establish your transfection protocol. But basically, last transfection reagents are ok to make tests.

for that point it depends on which interaction you wanna study.

hope that helps.
fred

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thanks for replying fred! it does help. another question is whether i need to used a certain kind of vector for transfecting primary cells. i was just going to use commercially available mammalian expression vector to express cos7 cells. can i still use that one or do i need to use, say, a retroviral vector? thank you.

s70048

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